Methods of receptor modulation and uses therefor

ABSTRACT

Receptor modulating agents capable of modulating cell surface receptors by affecting the cell surface receptor trafficking pathway are utilized for the treatment and diagnosis of a variety of disorders in warm-blooded animals, including neoplastic disorders. The receptor modulating agents are comprised of a covalently bound rerouting moiety and targeting moiety.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of U.S. patent applicationSer. No. 08/224,831, filed Apr. 8, 1994, now abandoned.

TECHNICAL FIELD

The present invention is generally directed to use of receptormodulating agents which modulate cell surface receptors and, morespecifically, to the use of receptor modulating agents which bind tocell surface receptors and affect the receptor trafficking pathway forthe treatment and diagnosis of a variety of disorders in warm-bloodedanimals.

BACKGROUND OF THE INVENTION

Cell surface receptors constitute a class of proteins which areresponsible for receptor-mediated endocytosis of specific ligands.Basically, the receptors serve as escorts for ligand delivery tointracellular destinations.

Ligand delivery is generally achieved through coated regions on theplasma membrane called "coated pits." These pits continually invaginateand pinch off, forming "coated vesicles" in the cytoplasm. Coated pitsand vesicles provide a pathway for receptor mediated endocytosis ofspecific ligands. The ligands that bind to specific cell surfacereceptors are internalized via coated pits, enabling cells to ingestlarge numbers of specific ligands without taking in correspondinglylarge volume of extracellular fluid. The internalized coated vesiclesmay or may not lose their coats and bind with other vesicles to formlarger vesicles called "endosomes." In the endosome the ligand and thereceptor are separated or "sorted." Endosomes which sort ligands andreceptors are known as "compartment of uncoupling of receptor andligand" or "CURL."

Endosomes may fuse with primary lysosomes, where their contents aredigested, or they may be delivered to other intracellular destinations.The receptor proteins are generally not digested, but are ratherrecycled to the cell membrane surface through a process called"exocytosis," or transferred to early or late endosomes viamultivesicular bodies. The entire pathway is referred to as the"receptor trafficking pathway."

Some receptors deliver their ligand directly to the cytoplasm or otherspecific intracellular locations. Perhaps one of the most studiedreceptor trafficking pathways is that of iron transport. In thispathway, a serum carrier protein, transferrin, binds iron and transportsit to transferrin receptors on the plasma membrane surface. Afterbinding and internalization, via coated pits, the resulting vesiclecombines first with early endosomes and then with late endosomes. Thisprocess results in the gradual drop in pH in the vesicle. The drop in pHcauses the transferrin carrier protein to lose its affinity to iron.When this occurs, the iron translocates through the membrane of thevesicle and joins the intracellular pool of enzymes. The transferrinreceptor may then recycle to the cell surface where it may repeat theprocess.

Other receptors may deliver their ligand directly to the lysosomes fordigestion. For example, the epidermal growth factor ("EGF") receptordelivers its ligand directly to a lysosome for degradation (Prog.Histochem. Cytochem. 6:39-48,1992). The EGF receptor may recycle to thecell surface depending on its state of phosphorylation (Cancer Treat.Rep. 61:139-160, 1992; J. Cell. Biol. 116:321-330, 1992).

A single receptor may utilize more than one receptor trafficking pathwaywithin the same cell. For example in polarized cells, such asspecialized transport epithelia cells, membrane trafficking is distinctbetween apical and basal sides of the cell (Sem. Cell. Biol. 2:387-396,1991). Moreover, non-polarized epithelia cells may simultaneously followtwo separate sorting pathways.

The control or regulation of cell surface receptors may be achieved by avariety of techniques. Regulation of cell surface receptors may beaccomplished, at a very basic level, by the binding of naturallyoccurring ligands. As discussed above, receptor binding of a ligand willgenerally trigger the internalization of the ligand-receptor complex.Such internalization may desensitize the cell to further ligand binding.(J. Immunol. 150:3161-9, 1993; Mol. Endocrinol. 6:2090-102, 1992; J.Cell. Physiol. 154:281-8, 1993; Receptor 1:13-32, 1990-91; Biochem. J.288:55-61, 1992; J. Immunol. 148:2709-11, 1992; J. Cell. Physiol.148:24-34, 1991). This type of regulation, however, is transient innature and does not result in diminution of biologic response.

Regulation of cell surface receptors may also be accomplished byadministration of receptor antagonists or agonists. Receptor antagonistsare organic protein or peptide ligands generally derived throughempirical structure-function studies, or through the use of detailedknowledge of ligand and receptor interaction. Essentially, an antagonistmay constitute any molecule with similar binding activity to a naturalligand, but incapable of producing the biological response normallyinduced by the natural ligand. Thus, the antagonist competitively blocksreceptor activity. With a competitive antagonist, the regulation ofreceptor activity is dependent upon both the antagonist's affinity forthe receptor, as well as its extracellular concentration over time.Receptor agonists are protein or peptide ligands derived in a similarmanner as antagonists. Essentially, an agonist may constitute anymolecule which binds to the receptor in a manner superior to that of thenatural ligand.

One receptor of particular interest is the vitamin B₁₂ receptor. As hasbeen demonstrated in experimental in vitro data, pre-clinical animalmodels, and patient studies, vitamin B₁₂ is a co-enzyme necessary incell division, as well as cellular metabolism, in proliferating normaland neoplastic cells. Insufficient vitamin B₁₂ causes cellular divisionto be held in abeyance and ultimately may result in apoptosis. Thenutrient is generally derived from dietary intake and is transportedthroughout the body complexed to transport proteins. The complex oftransport protein and vitamin B₁₂ is recognized by a cellular receptorwhich internalizes the complex and releases the vitamin intracellularly.The overall process has been reviewed in GUT 31:59, 1991. Vitamin B₁₂ istaken in through the diet. Binding proteins in the saliva (R-binder) andgut (intrinsic factor-(IF)) complex vitamin B₁₂ after release fromendogenous binding proteins by action of enzymes and low pH in thestomach. Vitamin B₁₂ is transferred across the intestinal epithelium ina receptor specific fashion to transcobalamin II (TcII). The vitamin B₁₂/transcobalamin II complex is then transported throughout the body andrecognized by receptors present on dividing cells, internalized andreleased within the cell where it is utilized by certain enzymes as aco-factor.

The high affinity receptor in dividing tissues or cells responsible forinternalization of vitamin B₁₂ recognizes transcobalamin II complexedwith vitamin B₁₂. The vitamin B₁₂ /TcII receptor recognizes only thevitamin B₁₂ /TcII complex and not the serum transport protein or thevitamin alone. The receptor is undetectable on non-dividing cells; themechanism for supplying non-dividing cells with vitamin B₁₂ is poorlyunderstood. However, it is known that more vitamin B₁₂ is requiredduring cell division than during metabolism, and that the vitamin B₁₂/TcII receptor is the only high affinity means for cellular uptake ofvitamin B₁₂ during cell division. When stimulated to divide, cellsdemonstrate transient expression of this receptor leading to vitamin B₁₂uptake which precedes actual DNA synthesis (J. Lab. Clin. Med. 103:70,1984). Vitamin B₁₂ receptor levels may be measured by binding of ⁵⁷Co-vitamin B₁₂ complexed to transcobalamin II (present in serum) onreplicate cultures grown in chemically defined medium without serum. Noreceptor mediated uptake occurs in the absence of carrier protein.

Dividing cells, induced to differentiate, lose receptor expression andno longer take up vitamin B₁₂. More importantly, leukemic cells,deprived of vitamin B₁₂, will stop dividing and die (Acta Haemat. 81:61,1989). In a typical experiment, leukemic cell cultures were deprived ofserum for 3 days, and then supplemented either with serum (a source ofvitamin B₁₂) or a non-metabolizable analogue of vitamin B₁₂ and culturedup to five days. Cell cultures supplemented with vitamin B₁₂ continuedto grow, whereas those deprived of the active nutrient stopped growingand die.

Based on these observations, it has been suggested that whole bodydeprivation of vitamin B₁₂ may be useful in the treatment of cancer orother disorders characterized by uncontrolled growth of cells. Moreover,because of the critical role played by vitamin B₁₂ -containing enzymesin cell division, it is believed that vitamin B₁₂ deprivation may beused in combination with chemotherapeutic drugs which inhibit cellularreplication. For example, when vitamin B₁₂ depletion was combined withmethotrexate, the two modalities together were more efficient indepleting folate levels in leukemic cells than either alone (FASEB J.4:1450, 1990; Arch. Biochem. Biophys. 270:729, 1989; Leukemia Research15:165, 1991). Folates are precursors in the production of DNA andproteins. In typical experiments, cultures of leukemic cells wereexposed to nitrous oxide for several hours to convert the active form ofendogenous vitamin B₁₂ to an inactive form. Replicate cultures were thenleft without further treatment, or additionally treated withmethotrexate. Cellular folate levels were measured three days later.Cells treated with the combination (i.e., both methotrexate and inactivevitamin B₁₂) showed a more striking decrease in cellular folate levelsthan with either of the two approaches alone. This combination alsoresults in a higher cell kill in vitro. When this approach was appliedto the treatment of highly aggressive leukemia/lymphoma in animal models(Am. J. Haematol. 34:128,1990; Anticancer Res. 6:737, 1986; CancerChemother. Pharmacol. 17:114, 1986; Br. J. Cancer 50:793, 1984),additive or synergy of anti-tumor action was observed, resulting inprolonged remissions and cures.

A key finding in the experiments described above was that short-term(hours to days), whole body depletion of vitamin B₁₂ can actsynergistically with chemotherapeutic drugs (such as methotrexate and5-FU) to inhibit tumor growth and treat animals with leukemia/lymphoma.Despite synergistic anti-tumor activity, there was no toxicityattributable to the short-term vitamin B₁₂ depletion for proliferatingnormal cells. This combination therapy was demonstrated in multipleanimal models. Observations in patients have indicated that long-term(months to years) vitamin B₁₂ depletion is required to producesignificant normal tissue toxicity. Even in those cases, subsequentinfusion of vitamin B₁₂ can readily reverse symptomology (Br. J. Cancer5:810, 1989).

Because of the promise of this therapeutic approach, various methodshave been sought to efficiently and controllably perform a temporarydepletion of vitamin B₁₂. Such methods, however, affect all of thebody's stores of vitamin B₁₂. They include dietary restriction, highdoses of vitamin B₁₂ analogues (non-metabolizable-competitiveantagonists which act as enzyme inhibitors), and nitrous oxide(transformation of vitamin B₁₂ to inactivate form). These differentmethods have been used in culture systems and in animals to depletevitamin B₁₂. The most efficient and the most utilized method has beenthe inhalation of nitrous oxide (laughing gas). Animals are maintainedtypically under an atmosphere of 50% to 70% of nitrous oxide for periodsfrom a few hours to a few days, causing the conversion of endogenousvitamin B₁₂ into an inactive form. This methodology has been utilized incombination with drugs for therapy of leukemia/lymphoma. A furthermethod for vitamin B₁₂ depletion involves infusion of anon-metabolizable analogue of vitamin B₁₂ which essentially dilutes outthe active form. This form of therapy is not specific for dividing cellsbut affects liver dependent metabolic processes. Another approachincludes restricting the dietary intake of vitamin B₁₂. This method,however, requires very long periods of dietary restriction and is offsetby hepatic storage of vitamin B₁₂. All of these methods suffer fromproblems of specificity, since they affect both vitamin B₁₂ -dependentgrowth as well as basal metabolism, and therefore are not particularlysuited to the development of anti-proliferative pharmaceutical products.

In view of the biological importance of cell surface receptors,receptor-controlling agents have emerged as a class of pharmaceuticaldrugs. Moreover, with the advent of genetic engineering for theisolation and amplification of genes for cell surface receptors, as wellas computer programs to model the interactions between ligands andreceptors (i. e., "rational" drug design), the production ofreceptor-controlling drugs has been significantly enhanced.

To date, many months or even years of scientific research, as well assignificant financial resources, are required to produce new receptorantagonists or agonists. To speed up this process, new screeningtechnologies have been developed which utilize peptide or antibodyrecombinant libraries (see, e.g., Gene 73:305, 1988; Proc. Nat. Acad.Sci. (USA) 87:6378, 1990; Biochromatography 5:22, 1990; ProteinEngineering 3:641, 1989). While library screening does not require thesame degree of knowledge of a specific receptor/ligand system, it doesinvolve an intensive screening effort utilizing functionalreceptor-specific assays. Moreover, the initial compounds identified bysuch screening programs are generally only precursors to the developmentof therapeutic products through more typical structure-functionalassessments.

While antagonists and agonists are generally capable of regulating abiological response, the surface receptors which bind such ligands arecontinually being re-expressed on the cell surface. Thus, effectiveregulation by antagonists or agonists must rely on a relatively high andsustained serum concentration in order to bind the new surface receptorscontinually being expressed on the cell surface.

Accordingly, there is a need in the art for agents which bind cellsurface receptors and thus regulate biological responses associatedtherewith, and which further effect normal cellular trafficking of thebound receptor. There is also a need in the art for agents which, whenbound by a cell surface receptor and internalized, promotes retention ofthe receptor within the cell. Moreover, there exists a need for methodsrelating to the administration of such agents to regulate a biologicalresponse. The present invention fulfills these needs and providesfurther related advantages.

SUMMARY OF THE INVENTION

Briefly, the present invention is generally directed to a method formodulating a cell receptor, comprising administering an effective amountof a receptor modulating agent to a warm-blooded animal such that areceptor is modulated. In a preferred embodiment, the receptormodulating agent utilized is comprised of a vitamin B₁₂ molecule coupledto a rerouting moiety.

In one embodiment of the present invention, the receptor modulatingagent utilized in the present invention is comprised of a reroutingmoiety coupled to a targeting moiety. Suitable rerouting moietiesinclude, by way of example, lysosomotropic moieties, such as gentamycin,kanamycin, neomycin, and streptomycin; intracellular polymerizingmoieties, such as dipeptide esters and leucine zippers; peptide sortingsequences, such as endoplasmic reticulum retention peptides, golgiretention peptides, lysosomal retention peptides, organism specificretention peptides and clathrin-binding peptides; conditional membranebinding peptides, such as charged glutamate, aspartate, and histidine;and bi- or multi-valent receptor cross-linking moieties. Suitabletargeting moieties include, by way of example, proteins, peptides, andnonproteinacious molecules, typified by antibodies, monoclonalantibodies.

Receptor modulating agents utilized in the present invention may act byaffecting a receptor trafficking pathway by redirecting anagent/receptor complex; by cross-linking one or more cell surfacereceptors; by anchoring a cell surface receptor in the membrane; byretaining a receptor in an endosome.

In a preferred embodiment of the present invention, a receptormodulating agent utilized in the present invention, is comprised of avitamin B₁₂ molecule coupled to a rerouting moiety. Suitable reroutingmoieties include, by way of example, lysosomotropic moieties, such asgentamycin, kanamycin, neomycin, and streptomycin; intracellularpolymerizing moieties, such as dipeptide esters and leucine zippers;peptide sorting sequences, such as endoplasmic reticulum retentionpeptides, golgi retention peptides, lysosomal retention peptides,organism specific retention peptides and clathrin-binding peptides;conditional membrane binding peptides, such as charged glutamate,aspartate, and histidine; and bi- or multi-valent receptor cross-linkingmoieties.

In another aspect of this preferred embodiment, the B₁₂ molecule iscoupled to the rerouting moiety by a linker. Generally, the linker is atleast 4 atoms in length, typically, the linker is about 6 to 20 atoms inlength and preferably, the linker is 12 atoms in length. Suitablelinkers include linkers which include an amino group, such asdiaminoalkyl, diaminoalkylaryl, diaminoheteroalkyl,diaminoheteroalkylaryl and diaminoalkanes. Preferably, the linker is--NH(CH₂)_(x) NH-- wherein x=2-20 or --NH(CH₂)_(y) CO--, wherein y=3-12.

In another aspect of this preferred embodiment of the present invention,the B₁₂ molecule is coupled to a rerouting moiety at a b-, d- or e-coupling site. In a particularly preferred embodiment of the presentinvention, the B₁₂ molecule is coupled to a rerouting moiety at a b-, d-and e- coupling site. In yet another embodiment, the B₁₂ molecule iscoupled to a rerouting moiety at a ribose coupling site. In yet anotheraspect of these embodiments, the receptor modulating agent is bound totranscobalamin.

In yet another preferred embodiment of the present invention, thereceptor modulating agent utilized is a vitamin B₁₂ dimer comprising afirst and a second vitamin B₁₂ molecule coupled through a coupling siteindependently selected from the group consisting of coupling sites a- tog-, coupling sites h, and coupling sites i. In a preferred embodiment,the B₁₂ molecule coupled through an e- or d- coupling site.

In another aspect of this embodiment, the B₁₂ molecules are coupled by alinker. Generally, the linker is at least 4 atoms in length, typically,the linker is about 10 to 55 atoms in length and preferably, the linkeris 35 to 45 atoms in length. In a preferred embodiment, the linker is atrifunctional linker. Suitable linkers include linkers which include anamino group, such as diaminoalkyl, diarninoalkylaryl,diaminoheteroalkyl, diaminoheteroalkylaryl and diaminoalkanes.Preferably, the linker is --NH(CH₂)_(x) NH-- wherein x=2-20 or--NH(CH₂)_(y) CO--, wherein y=3-12. In another aspect of thisembodiment, a dimer is coupled to at least one transcobalamin IImolecule.

In yet another embodiment, at least one of said first and said secondvitamin B₁₂ molecules of the dimer is a vitamin B₁₂ derivative.

Another embodiment of the present invention includes a method oftreating a neoplastic disorder, comprising administering atherapeutically effective amount of a receptor modulating agent to awarm-blooded animal suffering from a neoplastic disorder; said receptormodulating agent comprising a vitamin B₁₂ molecule coupled to arerouting moiety. Examples of neoplastic disorders include, leukemia,sarcoma, myeloma, carcinoma, neuroma, melanoma, cancers of the liver,lung, breast, brain, colon, cervix, prostrate, Hodgkin's disease, andnon-Hodgkin's lymphoma.

Yet another embodiment of the present invention includes a method forregulating a biological response associated with a cell surfacereceptor, comprising administering an effective amount of a receptormodulating agent to a warm-blooded animal such that a biologicalresponse is regulated.

These and other aspects of the present invention will become evidentupon reference to the following detailed description and attacheddrawings. In addition, various references set forth below which describecertain procedures or compositions in more detail are incorporated byreference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustrating a mechanism of action of a receptormodulating agent of the present invention. A healthy receptor willinternalize when bound by the appropriate ligand, release the ligandwithin the cell and then recycle to the cell surface. Receptormodulating agents of the present invention impede the receptortrafficking pathway by inhibiting the recycling of receptors to the cellsurface. Essentially, the targeting moiety on receptor modulating agentsbind the receptor and the rerouting moiety redirects thereceptor/receptor modulating agent complex to other points within thecell, where it may be retained or degraded. (Not shown in this schematicare receptors synthesized de novo).

FIGS. 2-5 are formulae representing families of antibiotics which act asrerouting moieties. The preferred reactive groups for coupling with atargeting moiety are indicated. These rerouting moieties facilitateretention of the receptor/receptor modulating agent complex throughprotonation of the complex, eventually delivering it to lysosomes fordegradation.

FIG. 2 consists of formulae representing the gentamycin, sisomicin, andnetilmicin families of antibiotics.

FIG. 3 consists of formulae representing the kanomycin, tobramycin, andamikacin families of antibiotics.

FIG. 4 consists of formulae representing the neomycin, paromomycin,ribostamycin, and butirosin families of antibiotics.

FIG. 5 consists of formulae representing the streptomycin family ofantibiotics.

FIG. 6 consists of formulae representing substituted aminoquinolines(e.g., chloroquine) substituted aminoacridines (e.g., quinacrine), andsubstituted aminonapthalines (e.g., dansyl cadaverine), all of which arerepresentative rerouting moieties of the present invention. Thesererouting moieties impede the receptor trafficking pathway throughprotonation and intracellular retention.

FIG. 7 consists of formulae representing glycosylation inhibitors, allof which are representative rerouting moieties of the present invention.These sugars may be conjugated to targeting moieties using linkagestypical of oligomeric carbohydrate chains. The resulting receptormodulating agent is recognized by internal glycosyl transferases,subject to intracellular retention, and, ultimately, degradation in thelysosomes.

FIG. 8 consists of a formula representing a vitamin B₁₂ (cyanocobalamin)molecule and identifies a preferred coupling site suitable for use inthe present invention for derivatization and conjugation.

FIG. 9 is a schematic depicting a representative reaction scheme for thesynthesis of a vitamin B₁₂ -GABA adduct.

FIG. 10a is a schematic depicting a representative reaction scheme forthe synthesis of a vitamin B₁₂ derivative comprising a vitamin B₁₂molecule with a diaminododecane linker arm coupled to any one ofcoupling sites d-, e-, or b-.

FIG. 10b is a schematic depicting a representative reaction scheme forcoupling a succinic anhydride to a vitamin B₁₂ diaminododecane adduct inpreparation for coupling the adduct to a rerouting moiety, or othermolecule, with an amino reaction site.

FIG. 11 is a schematic depicting a representative reaction scheme forthe synthesis of a vitamin B₁₂ derivative comprising a vitamin B₁₂molecule and a diaminododecane linker arm coupled to a ribose couplingsite.

FIG. 12 is a schematic depicting a representative reaction scheme forcoupling vitamin B₁₂ or a vitamin B₁₂ -GABA adduct to amikacin.

FIG. 13 is a schematic depicting a representative reaction scheme forcoupling vitamin B₁₂ or a vitamin B₁₂ -GABA adduct to streptomycin.

FIG. 14 is a schematic depicting a representative reaction scheme forcoupling a vitamin B₁₂ carboxylate derivative or a vitamin B₁₂ -GABAadduct to acridine.

FIG. 15 is a schematic depicting a representative reaction scheme forthe synthesis of a bivalent receptor modulating agent, a vitamin B₁₂dimer, using a trifunctional linker. The trifunctional linker allows forcoupling with additional compounds (R--NH₂) such as, by way of example,aminoglucosides (FIGS. 2-5), aminoacridines (FIG. 6), glycosylationinhibitors (FIG. 7), and biotin.

FIG. 16 is a schematic depicting a representative reaction scheme forthe synthesis of a vitamin B₁₂ dimer using a homobifunctional orhomotriflnctional cross-linking reagent.

FIG. 17 is a schematic depicting a representative reaction scheme forthe synthesis of a vitamin B₁₂ dimer using a heterobifunctionalcross-linker.

FIGS. 18-21 are schematics depicting representative reaction schemes forthe synthesis of various receptor modulating agents generally comprisedof a rerouting moiety, designated by the reactive group and R, selectedfrom those represented in FIGS. 2-7, and a vitamin B₁₂ molecule orderivative thereof as a targeting moiety.

FIG. 22 is a graph illustrating the binding curve of Transcobalamin IIto the cyanocobalamin monocarboxylic acids produced in Example 1.AD=Cyanocobalamin (1); AL=Cyanocobalamin b-monocarboxylic acid (2);AM=Cyanocobalamin e-monocarboxylic acid (3); and AN=Cyanocobalamind-monocarboxylic acid (4).

FIG. 23 is a graph illustrating the binding curve of Transcobalamin IIto the cyanocobalamin diaminododecane adducts produced in Example 3 and4. AH=Cyanocobalamin b-monocarboxylic acid conj Diaminododecane (7);AI=Cyanocobalamin e-monocarboxylic acid conj Diaminododecane (8);AJ=Cyanocobalamin d-monocarboxylic acid conj Diaminododecane (9);AK=Cobalamin e-monocarboxylic acid conj Diaminododecane, andAE=Cyanocobalamin Ribose-Succinate (11).

FIG. 24 is a graph illustrating the binding curve of Transcobalamin IIto a series of vitamin B₁₂ dimers. Dimer X=b-acid dimer withIsophthaloyl dichloride (36); Dimer Y=e-acid dimer with Isophthaloyldichloride (37); dimer Z=d-acid dimer with Isophthaloyl dichloride (38);Dimer A=b-acid Dimer with p-Iodo benzoyl Isophthaloyl dichloride (58);Dimer B=e-acid Dimer with p-Iodo benzoyl Isophthaloyl dichloride (59);and Dimer C=d-acid Dimer with p-Iodo benzoyl Isophthaloyl dichloride(60). These dimers were prepared as set forth in the Examples below.(see Examples 13 and 16.)

FIG. 25 is a graph illustrating the binding curve of Transcobalamin IIto a series of biotinylated vitamin B₁₂ molecules. AA=Cyanocobalaminb-monocarboxylic acid conj Diaminododecane and Biotin (17);AB=Cyanocobalamin e-monocarboxylic acid conj Diaminododecane and Biotin(18); AC=Cyanocobalamin d-monocarboxylic acid conj Diaminododecane andBiotin (19); AF=Cyanocobalamin Ribose-Succinate conj Diaminododecane(13); and AG=Cyanocobalamin Ribose-Succinate conj. Diaminododecane andBiotin (20). These biotinylated molecules were prepared as set forth inExamples below. (see Examples 8.)

DETAILED DESCRIPTION OF THE INVENTION

The present invention is generally directed to a receptor modulatingagent which is capable of binding to a cell surface receptor to form areceptor modulating agent/receptor complex ("agent/receptor complex").The binding of a suitable receptor modulating agent to a cell surfacereceptor generally results in invagination of the agent/receptor complexinto the cell into the vesicular system in the same manner as thenatural ligand. However, once internalized, or as part of theinternalization process, a receptor modulating agent of the presentinvention affects the receptor trafficking pathway by effectivelyimpeding, preventing, or delaying the receptor from recycling to thesurface, thus depriving the cell of receptors able to engage in bindingits natural ligand and triggering related biological responses.

Within the context of the present invention, "affecting the receptortrafficking pathway" refers to impeding the receptor trafficking pathwayin such a manner so as to affect biological response. This would includetrapping, delaying, retaining, re-directing, or degrading the cellsurface receptor. A "receptor modulating agent" is comprised of at leastone targeting moiety covalently attached to at least one reroutingmoiety. A "targeting moiety" is a moiety capable of specifically bindingto a cell surface receptor to yield an agent/receptor complex and, in apreferred embodiment, has an affinity for the cell surface receptor ofwithin 100-fold, and more preferably, within 10-fold, of the affinity ofthe natural ligand for the receptor. A preferred targeting moiety is avitamin B₁₂ molecule. In contrast, a "rerouting moiety" is a moietywhich redirects an agent/receptor complex, resulting in prolongedretention, degradation, and/or modulation of the receptor within theinterior of a cell or on the cell surface, including, by way of example,retaining the receptor in the cell membrane or directing the receptor toa lysosome within the cell. Suitable rerouting moieties are described indetail below.

A targeting moiety is coupled to a rerouting moiety to yield thereceptor modulating agent by any suitable means known in the art,including direct covalent linkage of an appropriate chemical linker orthrough a very tight association in non-covalent attachment. By way ofexample for the latter, in one embodiment, coupling is accomplishedthrough the combination of an avidin or streptavidin conjugate with avitamin B₁₂ /biotin conjugate. Coupling of the targeting moiety and thererouting moiety should be of a nature which resists cleavage by theenzymatic and low pH conditions normally encountered within the internalportion of the cell, including endosomes and lysosomes. Suitable linkersare noted below. The ability to resist cleavage may be detected by anymeans known in the art, including exposing the receptor modulating agentto enzymes at low pH and measuring release of the targeting or reroutingmoiety using techniques known in the art.

Coupling of a targeting moiety and a rerouting moiety should notsignificantly hinder the ability of the targeting moiety to specificallybind the cell surface receptor. The receptor modulating agent may alsoinclude additional moieties, so long as they do not interfere witheither the targeting or the rerouting moieties. For example, suchmoieties may be coupled to the receptor modulating agent through the useof a trifunctional linker or they may be coupled to a rerouting ortargeting moiety. Optimal attachment of the two moieties may bedetermined by comparing the affinity of binding of the receptormodulating agent with free targeting moiety in assays of inhibition ofbinding.

The binding specificity of a receptor modulating agent of the presentinvention via the targeting moiety can be determined using any one ofseveral methods known in the art, including competition assays against anatural ligand or against the targeting moiety itself in the absence ofthe remainder of the molecule. These, and other suitable techniques, aredescribed in detail in Sambrook et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor, 1989.

Covalent attachment of the targeting moiety and the rerouting moietyshould also not significantly affect the ability of the rerouting moietyto retain or delay the agent/receptor complex within the cell. This maybe empirically determined by any one of several methods known in theart, including using labeling techniques to compare intracellularretention of the targeting moiety versus that of the receptor modulatingagent as exemplified below.

As noted above, targeting moieties of the receptor modulating agentinclude any moiety which specifically binds to a cell surface receptor.Suitable targeting moieties include proteins, peptides, andnon-proteinaceous molecules. Representative examples of suitabletargeting moieties include antibody and antibody fragments; peptidessuch as bombesin, gastrin-releasing peptide, cell adhesion peptides,substance P, neuromedin-B, neuromedin-C and metenkephalin; hormones,including EGF, alpha- and beta-TGF, estradiol, neurotensin, melanocytestimulating hormone, follicle stimulating hormone, luteinizing hormone,and human growth hormone; proteins corresponding to ligands for knowncell surface receptors, including low density lipoproteins, transferrinand insulin; fibrinolytic enzymes; and biological response modifiers,including interleukin, interferon, erythropoietin and colony stimulatingfactor also constitute targeting moieties of this invention. Moreover,analogs of the above targeting moieties that retain the ability tospecifically bind to a cell surface receptor are suitable targetingmoieties. Essentially, any analog having about the same affinity as atargeting moiety, herein specified, could be used in synthesis ofreceptor modulating agents.

In a preferred embodiment, a targeting moiety is a vitamin B₁₂ molecule.Vitamin B₁₂ has a binding specificity of 2×10⁻¹¹ M. Vitamin B₁₂ is anessential nutrient for dividing cells. By inhibiting its uptake, thegrowth of dividing cells can be halted. The cell surface receptor forvitamin B₁₂ is the transcobalamin II/vitamin B₁₂ ("TcII/B₁₂ ") receptor,which is characterized by a high affinity for the carrier protein,transcobalamin II (TcII), when complexed with vitamin B₁₂ ("TcII/B₁₂complex"). The TcII/B₁₂ receptor does not recognize vitamin B₁₂ alone,but does recognize the carrier protein TcII with reduced affinity whennot complexed with vitamin B₁₂. In many respects, this receptor systemis similar to that for transferrin/iron in that the goal of the receptorsystem is to deliver vitamin B₁₂ into cells such that it can be utilizedby enzymes involved in DNA synthesis. Within the context of the presentinvention, the term "vitamin B₁₂ " refers to the class of compoundsknown as cobalamins and derivatives thereof, including, by way ofexample, cyanocobalamin. The term "vitamin B₁₂ " is used interchangeablywith the term cyanocobalamin.

Suitable vitamin B₁₂ molecules includes any vitamin B₁₂ capable ofcoupling to another molecule while maintaining its ability to form aTcII/B₁₂ complex. A preferred vitamin B₁₂ targeting moiety is generallycomprised of a vitamin B₁₂ molecule, such as a cyanocobalamin, and alinker, described in detail below. The linker may be coupled to any oneof several sites on a vitamin B₁₂ molecule, including potential carboxylcoupling sites a- through g-, an alcohol (ribose) coupling site("coupling site h") or a benzimidazole coupling site ("coupling sitei.") (See structure I below.) Preferably, a linker is coupled tocoupling sites b-, d- or e- on a vitamin B₁₂ molecule. Even morepreferably, a linker is coupled to coupling site d- or e-. Thisembodiment of the present invention includes compounds represented bythe following formula: ##STR1## wherein at least one of R₁, R₂, R₃, R₄,R₅, R₆, and R₇ is a linker. One of ordinary skill in the art willappreciate that a number of other coupling sites on the vitamin B₁₂molecule may be chemically altered without affecting coupling of themolecule with a linker or TcII. Coupling sites which are not occupied bya linker may have a variety of chemical moieties attached thereto,including an amino, secondary amino, tertiary amino, hydroxy, loweralkyl, lower alkoxy, alkoxyalkyl, alkoxyalkoxy, cycloalkylalkoxy, andthioalkyl groups.

In a preferred embodiment, R₁, R₂ or R₄ is a linker and the remaining Rgroups are --NH₂, with the exception of R₇, which is preferably --OH. Inan especially preferred embodiment, R₂ is a linker, R₁, R₃ -R₆ are --NH₂and R₇ is --OH.

In another preferred embodiment, R₇ is a linker and R₁ -R₆ are --NH₂.

                                      TABLE 1    __________________________________________________________________________    HOMOBIFUNCTIONAL LINKERS    __________________________________________________________________________     ##STR2##                                     disuccinimidyl suberate                                                  (DSS)*     ##STR3##                                     bis(sulfosuccinimidyl)                                                  suberate (BS.sup.3)*     ##STR4##                                     disuccinimidyl suberate                                                  (DSS)*     ##STR5##                                     bis(sulfosuccinimidyl)                                                  suberate (BS.sup.3)*     ##STR6##                                     disuccinimidyl tartarate                                                  (DST)*     ##STR7##                                     disulfosuccinimidyl                                                  tartarate (Sulfo-DST)*     ##STR8##                                     bis 2-(succinimidooxycarbony                                                  loxy)- ethyl!-sulfone                                                  BSOCOES)*     ##STR9##                                     bis 2-(sulfosuccinimidooxyca                                                  rbonyl- oxy)-ethyl!sulfone                                                  (Sulfo-BSOCOES)*     ##STR10##                                    bismaleimidohexane (BMH)*     ##STR11##                                    1,5-Difluoro-2,4-dinitrobenz                                                  ene (DFDNB)*     ##STR12##                                    dimethyl pimelimidate-2 HCl                                                  (DMA)*     ##STR13##                                    dimethyl pimelimidate-2 HCl                                                  (DMP)*     ##STR14##                                    dimethyl 3,3'-dithiobispropi                                                  onimidate-2 HCl (DTBP)*     ##STR15##                                    isophthalayl    __________________________________________________________________________                                                  dichloride**     *Pierce Chemical, Co., Rockford, Illinois     **Aldrich Chemical Co., Milwaukee, Wisconsin

                                      TABLE 2    __________________________________________________________________________    HETEROBIFUNCTIONAL LINKERS    __________________________________________________________________________     ##STR16##                         N-succinimidyl-3-(2-pyridylthio)propion                                       ate (SPDP)*     ##STR17##                         succinimidyl 6 3(2-pyridyldithio)                                       propionamido! hexanoate (LC-SPDP)*     ##STR18##                         sulfosuccinimidyl 6- 3-(2-pyridyldithio                                       ) propionamido! hexanoate                                       (Sulfo-LC-SPDP)*     ##STR19##                         succinimidyl 4-(N-maleimidomethyl)cyclo                                       hexane-1- carboxylate (SMCC)*     ##STR20##                         sulfosuccinimidyl 4-(N-maleimidomethyl)                                       cyclo- hexane-1-carboxylate                                       (Sulfo-SMCC)*     ##STR21##                         m-maleimidobenzoyl-N-hydroxysuccinimide                                        ester (MBS)*     ##STR22##                         m-maleimidobenzoyl-N-hydroxysulfosuccin                                       imide ester (Sulfo-MBS)*     ##STR23##                         N-succinimidyl(4-iodoacetyl)aminobenzoa                                       te (SIAB)*     ##STR24##                         sulfosuccinimidyl(4-iodoacetyl)aminoben                                       zoate (Sulfo-SIAB)*     ##STR25##                         succinimidyl-4-(p-maleimidophenyl)butyr                                       ate (SMPB)*     ##STR26##                         sulfosuccinimidyl-4-(p-maleimidophenyl)                                       butyrate (Sulfo-SMPB)*    __________________________________________________________________________     *Pierce Chemical, Co., Rockford, Illinois

                                      TABLE 3    __________________________________________________________________________    TRIFUNCTIONAL LINKERS    __________________________________________________________________________     ##STR27##                              Derived from 5-amino isophthalic*                                            acid - unreported synthesis (D.                                            S. Wilbur,  D. K. Hamlin, UW)     ##STR28##                              Derived from 3,5-diamino-benzoic                                            acid* - unreported synthesis     ##STR29##                              5-(p-iodobenzoyl)amino-1,3-isophth                                            aloyl  ditertra-fluorophenyl                                            ester - unreported  synthesis (D.                                            S. Wilbur, D. K. Hamlin,                                            University of Washington)     ##STR30##                              5(p-tri-N-butylisomylbenzoyl)-amin                                            o-1,3-isophthaloyl ditchtrafluorop                                            henyl ester - unreported                                            synthesis (D. S. Wilbur, D. K.                                            Hamlin, UW)     ##STR31##                              synthesis as reported: D. S.                                            Wilbur et al., Bioconjugate Chem.                                            5(3):220--235, 1994.    __________________________________________________________________________     *Aldrich Chemical Co., Milwaukee, Wisconsin

Suitable linkers include any one of several linkers, preferablycontaining at least two coupling or reactive groups, allowing the linkerto bind to both vitamin B₁₂ and a rerouting moiety. In the context ofthe present invention, the terms "coupling group" and "reactive group"are used interchangeably. By way of example, a linker may behomobifunctional, heterobifunctional, homotrifunctional, orheterotrifunctional. Homobifunctional agents may facilitatecross-linking, or dimerization of vitamin B₁₂ molecules in a singlestep, hence a coupling reaction using these agents should be performedwith an excess of homobifunctional agents, unless dimerization is thedesired result, as in the synthesis of dimers described in detail below.Suitable homobifunctional agents include those listed in Table 1, aswell as those described in detail below. Heterobifunctional agentsfacilitate cross-linking in a stepwise method, allowing more than onelinker to be incorporated and a variety of targeting agents such asvitamin B₁₂ molecules to be linked. Suitable heterobifunctional agentsinclude those listed in Table 2 as well as those described in detailbelow. Homo- and hetero-trifunctional linkers are coupled to a reroutingmoiety and a vitamin B₁₂ molecule as described above, with theadditional advantage of a third coupling site on the linker. One ofordinary skill in the art will appreciate that this allows for anynumber of different molecules to couple with the rerouting moiety,including, by way of example, markers, such as radiolabeled andfluorescent molecules; proteins and peptides, such as antibodies; andconjugating molecules, such as biotin. Suitable trifunctional linkersare listed in Table 3. Homobifunctional, heterobifunctional,homotrifunctional, and heterotrifunctional linkers are commerciallyavailable.

Suitable linkers are generally relatively linear molecules greater than4 atoms in length, typically between 6 and 30 atoms in length, andpreferably are 8 to 20 atoms in length. In a particularly preferredembodiment, the linker is a linear molecule of 12 atoms in length. Inthe context of the present invention, the term "atom" refers to achemical element such as, by way of example, C, N, O, or S. The rangesprovided above are based on the relatively linear accounting of thelinker. One of ordinary skill in the art will appreciate that a linkermay be linear, branched, and even contain cyclical elements.

Coupling or reactive groups include any functional group capable ofcoupling a linker to a vitamin B₁₂ molecule. Suitable coupling groupsinclude, nucleophilic and electrophilic functional groups. Suitablenucleophilic groups include hydroxy groups, amino groups, and thiogroups. Suitable electrophilic groups include carboxylic acid groups andcarboxylic acid derivatives including acid halides, acid anhydrides, andactive esters such as NHS esters.

Suitable homobifunctional linkers include, by way of example,diaminoalkanes, such as those represented by the formula NH₂ (CH₂)_(x)NH₂, wherein x=2-20. A preferred linker is a diaminododecane. Suitableheterobifunctional linkers include those represented by the formula NH₂(CH₂)_(y) COOH, wherein y=3-12. Those of ordinary skill in the art willappreciate that a protecting group may be necessary when utilizing aheterobifinctional group.

A linker may be coupled to the preferred b-, d- or e- coupling sites(see Structure I above) by any one of several suitable means, including,by way of example, activating a vitamin B₁₂ molecule by hydrolyzing itspropionamide groups to produce monocarboxylates, purifying the resultingmonocarboxylates, and coupling a linker to a selected coupling site.Hydrolysis of the coupling sites may be accomplished by exposing vitaminB₁₂ to aqueous acid for a period of time and under suitable conditionsto hydrolyze the desired propionamide groups. Preferably, hydrolysis isperformed by exposure of the amide to dilute aqueous acid for a periodof about 6 to 12 days, typically about 9 to 11 days, and most preferablyabout 10 days at room temperature. Suitable aqueous acids include, byway of example, 0.1N hydrochloric acid, 0.5N phosphoric acid or 0.5Nsulfuric acid.

Purification of b-, d- and e- monocarboxylates can be accomplished byany one of several means, including column chromatography, such as gelpermeation chromatography, adsorption chromatography, partitionchromatography, ion exchange chromatography, and reverse phasechromatography. Preferably, column chromatography is preparative reversephase liquid chromatography. These techniques are described in detail inLim, HPLC of Small Molecules, IRL Press, Washington, D.C., 1986.Purification of monocarboxylates by preparative liquid chromatography(LC) should be accomplished at a very slow flow rate. For example, LCpurification may be conducted at a flow rate of 0.15 mL/min. on a 5 μm,4.6×250 mm propylamine column (RAININ microsorb-MV amino column) elutingwith 58 μM pyridine acetate, pH 4.4 in H₂ O:THF (96:4) solution. Evenmore preferably, the coupling reaction is monitored using analyticalhigh pressure liquid chromatography (HPLC). Reverse-phase HPLCchromatography is preferably carried out using an analytical version ofabove-noted propylamine column using a gradient solvent system at a flowrate of 1 mL/min. Within the context of the present invention, the d-isomer is identified as the longest retained peak (third), the e- isomeris identified as the second retained peak, and the b- isomer isidentified as the shortest retained peak (first) eluted from the LCcolumn. The d- isomer may also be identified as that vitamin B₁₂derivative demonstrating the greatest biological activity as notedbelow.

A ribose coupling site (coupling site h, see structure I) may beactivated by any one of several suitable means including, activating ahydroxyl group at coupling site h by reaction with a suitable reagent(e.g., succinic anhydride), to yield a ribose derivative which bears areactive group (e.g., a carboxylate group). This technique is describedin detail in Toraya, Bioinorg. Chem. 4:245-255, 1975. Separation andpurification of the activated molecule may be accomplished on C1 8column as noted above. Once coupling site h has been activated, a linkermay be coupled to this site in the same manner as described below.

After activating the vitamin B₁₂ molecule at a selected coupling site,linkers may be coupled to a vitamin B₁₂ molecule to form a vitamin B₁₂linker adduct using any one of several means, including, by way ofexample, an amide forming reaction, employing an amine group on thelinker and a carboxylate coupling site on a vitamin B₁₂ molecule.Alternatively, a linker may be coupled to a vitamin B₁₂ molecule throughan amide forming reaction, employing a carboxylate group on the linkerand an amino group on a B₁₂ molecule. The amide forming reaction mayinclude the use of a coupling agent. Suitable coupling agents includecarbodiimide coupling agents, such as, by way of example,1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC),1-benzyl-3-(3-dimethylaminopropyl) carbodiimide (BDC),1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide (CMC), and1,3-dicyclohexylcarbodiimide (DCC). Preferably, the coupling agent iswater soluble. Even more preferably, the coupling agent is EDC.

Alternatively, the amide forming reaction coupling the linker to a B₁₂molecule may employ a reactive carboxylic acid group and an amine.Suitable reactive carboxylic acid groups include carboxylic acidderivatives which yield an amide upon reaction with an amine. Suchreactive groups include, by way of example, any reactive carboxylic acidderivative, including, by way of example, carboxylic acid halides, suchas acid chlorides and bromides; carboxylic acid anhydrides, such asacetic anhydrides and trifluoroacetic anhydrides; esters, such asp-nitrophenyl esters and N-hydroxysuccinimide esters. Such techniquesare described in detail in Bodanszky, Principles of Peptide Synthesis,Springer Verlag, Berlin, 1984.

Although coupling of a linker through a cyano coupling site is possibleit is not preferred, due to the instability of linkers coupled to thissite. Dolphin, D., 205! Methods Enzymol. 18C:34-52, 1971. Additionally,a linker may be coupled to a benzimidazole (coupling site i, seeStructure I) using techniques described in detail in Jacobsen, Anal.Biochem. 113:164-171, 1981.

Vitamin B₁₂ linker adducts may be separated and purified using anysuitable means, including column chromatography, such as gel permeationchromatography, adsorption chromatography, partition chromatography, ionexchange chromatography, and reverse phase chromatography. Preferably,column chromatography is preparative LC. These techniques are describedin detail in Lim, HPLC of Small Molecules, IRL Press, Washington, D.C.,1986.

As noted above, the vitamin B₁₂ receptor modulating agents of thepresent invention must be capable of binding transcobalamin II. Theability of a receptor modulating agent to bind TcII may be ascertainedusing any one of several means known in the art, including competitivebinding assays with the receptor modulating agent competing with nativevitamin B₁₂.

In another embodiment of this invention, a targeting moiety is anantibody. In the context of the present invention, the term "antibody"includes both monoclonal and polyclonal antibodies and further includesan intact molecule, a fragment thereof, and a functional equivalentthereof. Particularly preferred antibodies include monoclonal antibodieshaving high specificity for a cell surface receptor and the ability toprovoke the internalization of a target receptor. Suitable antibodiesmay be selected by assays for internalization known in the art anddescribed in detail in Cancer Treat. Res. 68:23, 1993; Leuk. Lymp.9:293, 1993; Anticancer Drug Des. 7:427, 1992.

Despite the potential utility of antibodies and antibody derivatives astargeting moieties, there may be pharmaceutical applications for whichthey are not appropriate due to their cost, potential forimmunogenicity, or need for specialized forms of delivery such asorthotopic or oral administration. For these purposes, small organiccompounds or peptides may be more suitable. Such peptides and compoundsmay be isolated by: (1) screening of bacterial peptide expressionlibraries, antibody paratope analogs or antibody Fab expressionlibraries to identify peptide or antibody variable region inhibitors(Gene 73:305, 1988; Proc. Nat. Acad. Sci. USA 87:6378, 1990;BioChromatography 5:22, 1990; Protein Engineering 3:641, 1989); (2)rational drug design programs using antibodies as a "pharmacophore" tocreate organic molecule analogs (Biotechnology. Jan. 19, 1991), ortraditional rational drug design programs using crystallized vitaminreceptor to identify peptide or organic inhibitors (Biochem. J. 268:249,1990; Science 248:1544, 1990); and (3) screening a library of organicmolecules, as present in fermentation broths of microorganisms, forinhibition of vitamin B₁₂ uptake, identifying the biochemical nature ofinhibitory compound(s), and chemically synthesizing analogs to explorestructure-finction relationship and to identify potent inhibitor(s).

Small organic compounds and peptide receptor antagonists for the B₁₂receptor may be identified through the use of an appropriate assay. Forexample, specific binding assays using antibodies which act ascompetitive antagonists. Through these means a repertoire of protein andnon-protein molecules suitable for human use can be generated, and maybe used to define optimal regimens to manipulate vitamin B₁₂ uptake andbioavailability for different pharmaceutical applications that requirean alteration in cellular proliferation.

Rerouting moieties of the present invention include any moiety which iscapable of affecting the receptor trafficking pathway. Thischaracteristic can be assessed by employing a receptor modulating agenthaving a radiolabeled targeting moiety and following its path throughthe cell. This is accomplished using techniques known in the art,including using radiolabeled, biotinylated, or FITC labeled targetingmoiety followed by binding assays, ELISA, or flow cytometry. A preferredreceptor modulating agent is one which results in the removal of thehighest percent of receptor for the longest period of time.

Suitable rerouting moieties of this invention do not significantlydetract from the selectivity of the targeting moiety. Whether arerouting moiety detracts from the selectivity of a targeting moiety maybe determined by any one of several methods known in the art, includingcomparing binding of the receptor modulating agent on receptor positiveand receptor negative cells, as assessed by ELISA, flow cytometry, orother binding assays.

Rerouting moieties cause the retention/degradation of an agent/receptorcomplex within at least one cell type, but not necessarily in all cells.In like fashion, a rerouting moiety causes retention of anagent/receptor complex in some cells, but not necessarily otheragent/receptor complexes in other cells. Different rerouting moietiesmay also distinguish between receptor species, for example, as inpolarized epithelium where the same receptor may independently trafficon the apical, basal, or basolateral sides of the cell. To determine ifa particular rerouting moiety is suitable, a rerouting moiety iscovalently attached to the targeting moiety, and the resulting receptormodulating agent is compared for receptor modulation on differentreceptor-bearing cells using binding or functional assays known in theart.

Suitable rerouting moieties of this invention may be categorized intofive different functional classes: (1) lysosmotropic moieties; (2)intracellular polymerizing moieties; (3) protein sorting signals orsequences; (4) conditional membrane binding peptides; and (5) bi- ormulti-valent receptor cross linking moieties. While such reroutingmoieties may have different functional mechanisms of action, all promoteretention of the agent/receptor complex within the intracellularvesicular system. All of these classes of rerouting moieties will impartthe ability to affect the receptor trafficking pathway.

In one aspect of the present invention, a first functional class ofrerouting moieties, lysosomotropic moieties, are disclosed. Within thecontext of the present invention, the term "lysosomotropic moieties"refers to moieties which route the agent/receptor complex to thelysosomes. Numerous suitable lysosomotropic moieties are known, and arereviewed in Biochem. Pharmacol. 23:2495-2531, 1974.

A preferred lysosomotropic moiety includes an aminoglycoside antibioticmarked by the characteristic ability to accumulate in lysosomes afterintracellular protonation. Intracellular protonation occurs in theincreasingly acidic conditions which occur during the transfer fromearly to late endosomes and, finally, to the lysosome. Strong positivecharges prohibit the lysosomotropic moiety from leaving themembrane-enclosed vesicles, thus trapping the agent/receptor complex inthe vessel.

Aminoglycoside antibiotics are similar in structure, but are dividedinto structurally related families of compounds based upon the sugarunits. Each of the families of aminoglycoside antibiotics, as well asrepresentative members thereof, are set forth in FIGS. 2-5. Thesefamilies include gentamycin, kanamycin, neomycin and streptomycin. Thegentamycin family includes gentamycin C₁, gentamycin C₂, gentamycinC_(1a), sisomicin and netilmicin; the kanamycin family includeskanamycin A, tobramycin and amikacin; the neomycin family includesneomycin B, paromomycin, ribostamycin and bytirosin B; and thestreptomycin family includes streptomycin A and streptomycin B.

In a particularly preferred embodiment of the present invention, thererouting moiety is gentamycin, which accumulates in lysosomes inconcentration as much as 300 fold that of the extracellularconcentration (J. Pharmacol. Exp. Ther. 255:867-74, 1990; Ren. Fail.14:351-7, 1992).

Suitable aminoglycosides have reactive amine groups capable of beingcoupled through peptide or other chemical linkers. Thus, a targetingmoiety may be readily attached via covalent linkage to these reroutingmoieties using any one of several techniques known in the art to formcovalent bonds, for example, using thioether, disulfide, ether, esterand peptide bonds. Since many of the aminoglycoside antibiotics haveseveral amines which could be derivatized in a conjugation procedure, aprimary amine contained in these compounds can be selectively reacted tofavor covalently attachment to the targeting moiety through this amine(see amine indicated with arrow in FIGS. 2-4). With regard tostreptomycin, covalent attachment to the targeting moiety may beaccomplished by converting the aldehyde moiety indicated in FIG. 5 to anamine, and attaching to the targeting moiety using carbodiimide or othersuitable activated carboxylic acid. Aminoglycosides are water solubleand do not readily bind to other proteins, and thus do not impartnon-specific binding to a receptor modulating agent.

Particularly preferred aminoglycosides include those which allow forpreferential derivation of a selected amine. Specifically, preferredaminoglycosides include those compounds to which protective groups canbe added to various nitrogen atoms thereof and, subsequently,selectively deprotected to yield a single free amine. The free amine canbe further derivatized, for example, by addition of a peptide linker orcovalently attached directly to the targeting moiety. These reroutingmoieties include ribostamycin (see FIG. 4), kanamycin (see FIG. 3),amikacin, and streptomycin. Ribostamycin is particularly preferred, dueto its relative low toxicity and its derivatization chemistry, allowingan acyl migration reaction to be effected on a hydroxyl protectedribostamycin to yield a single amine adduct. Kanamycin may also be usedin a selective protection/acylation reaction; Amikacin is commerciallyavailable in a form which allows attachment without deprotecting itsamines or alcohol groups; and streptomycin can also be readilyderivatized by protonating guanidinium groups under physiologicconditions to provide the polycations necessary for cellular orlysosomal retention.

In another aspect of the present invention, non-aminoglycosidelysosomotropic compounds which may accumulate after intracellularprotonation are also suitable rerouting moieties (see FIG. 6). Suitablenon-aminoglycoside compounds exhibiting this characteristic are known inthe art, a series of aminoacridine and amino quinoline dyes, typified bycholquinine and quinacrine; a group of amino naphthalenes, typified bydansyl cadaverine; and derivatives thereof. Such dyes are characterizedby cellular retention and low toxicity. All of these compounds havecharacteristic sites for covalent attachment to a targeting moiety viathe nitrogen indicated in FIG. 6 and may be attached thereto asdescribed above.

Another aspect of the present invention utilizes a lysosomotropicpeptide subject to charge modification under intracellular conditions isemployed as a rerouting moiety. Once charge-modified, the reroutingpeptide acts to retain an agent/receptor complex in the intracellularvesicular system until membrane flow delivers it to the lysosome fordegradation. Preferably, these peptides are capable of beingphosphorylated by intracellular protein kinases. When phosphorylated bythe intracellular enzymes, such peptides would be highly anionic.

Charge-based retention can be an inherent property of the reroutingpeptide or can be imparted by intracellular modification. Intracellularmodification may be accomplished by any of several means known in theart, including phosphorylation of certain residues of some receptors(e.g., the EGF receptor) may cause intracellular rerouting (CancerTreat. Res. 61:139-160, 1992; J. Cell. Biol. 116:321-30, 1992).

The rerouting peptides may be covalently attached to a targeting moietyby any means, including, for example, covalently linking the peptidedirectly to the targeting moiety, or by use of an appropriate linkermoiety, such as G-G-G, which may be derivatized and covalently attachedto the targeting moiety.

Preferred rerouting peptides include protein kinase-substrate peptidesthat incorporate serine. These peptides are particularly preferred forenhancement of receptor rerouting in tumor target cells, which haveincreased levels of protein kinase activity for serines or tyrosines.Increased levels of kinase activity within tumor cells may be attributedto the presence of oncogene products, such as H-ras, on the cytoplasmicside of tumor cell plasma membranes (C.I.B.A. Found. Symp. 164:208-18,1992).

Suitable rerouting peptides also include protein kinase substrates andpeptides that possess a single positive charge. The latter type ofrerouting peptide may form an ion pair with a "glutamate-like" residueof an attached or closely associated residue(s) of the receptor.Particularly preferred rerouting peptides may be derived, usingtechnologies known in the art, from the proteins and the amino acidsequences identified in Table 4.

                  TABLE 4    ______________________________________    REROUTING PEPTIDES    PEPTIDE SOURCE    AMINO ACID SEQUENCE    ______________________________________    EGF receptor      DVVDADEYLIPQ    BGF fragment      CMHIESLDSYTC    Phosphorylase kinase                      RTKRSGSVYEPLKI    Protein kinase C pseudosubstrate                      RFARK-GALRQKNV    Myelin basic protein                      S/T-XAA-K/R (where XAA is                      an uncharged residue)    Kemptide          RGYALG or RGYSLG    Glycogen synthetase                      PLSRTLSVAA    Transferrin receptor                      FSLAR    III histone       ASGSFKL    Casein kinase II substrate                      AAAAAASEEE or                      AAAAAASDDD    Insulin receptor auto-phosphorylation                      DIYETDYYR    substrate    calmodulin-dependent protein kinase                      Waxman and Arenowski Biochem.    II                32(11):2923-30, 1993    Neurogranin       Chen et al., Biochem. 32(4):    MARCKS            1032-9, 1993                      Heemskerk et al., Biochem.                      Biophys. Res.                      Commun. 190(1):236-41, 1993    Glycogen synthase Marais et al., FEBS Letters                      277:151-5, 1990    Ribosomal protein S6                      Munro et al., Biochem. Biophys.                      Acta 1054:225-30, 1990    Co-polymers which serve as                      Abdel-Ghony et al., Proc.    substrates for protein kinase A, C, P                      Nat'l. Acad. Sci.                      86:1761-5, 1989; Abdel-Ghony                      et al., Proc. Nat'l. Acad. Sci.                      85:1408-11, 1988    Serine-threonine kinases                      Abdel-Ghony et al., Proc. Nat'l.                      Acad. Sci. 86:1761-5, 1989;                      Abdel-Ghony et al., Proc. Nat'l.                      Acad. Sci. 85:1408-11, 1988    ______________________________________

In another aspect of the present invention, the rerouting moiety is alysosomotropic amino acid ester which, in high concentration, can causethe lysis of granule containing cells, such as NK cells, cytolytic Tcells and monocytes. The concentration must generally be maintainedbelow 100 mM to avoid lysis. Suitable lysosomotropic amino acid estersand their sources are presented in Table 5.

                  TABLE 5    ______________________________________    LYSOSOMOTROPIC AMINO ACID ESTERS    ______________________________________    Leu--O--Me     Res. Immunol. 143:893-901, 1992                   Eur. J. Immunol. 23:562-5, 1993                   Intl. Arch. Aller. & Immunol. 100:56-59,                   1993                   Cell. Immunol. 139:281-91, 1992                   Exp. Pathol. 42:121-7, 1991    Iso--leu--O--Me                   Res. Immunol. 143:893-901, 1992    L--Val--O--Me  J. Immunol. 134:786-93, 1985    Phe--O--Me     J. Immunol. 148:3950-7, 1992                   Blood 79:964-71, 1992    Phe--, Ala--, Met--, Trp--,                   Int. J. Immunopharmacol. 13:401-9 1991    Cys--, Try--, Asp--, &    Glu--O--Me    ______________________________________

The lysosomotropic amino acid esters identified in Table 5 can be usedto retain the agent/receptor complex in lysosomes after intracellularcleavage of the ester. In one embodiment, such amino acid esters may beutilized as the C-terminal portion of a larger peptide containing alinker sequence and/or a phosphorylation substrate sequence, and withsuitable residues, such as cysteine, for covalent attachment to atargeting moiety, such as a sequence encoding a peptide or proteinligand for a given cell surface receptor.

In another embodiment of the present invention, a second functionalclass of rerouting moieties is disclosed. This class includes peptideswhich undergo polymerization within endosomes or lysosomes, inhibitingtheir passage through intracellular membranes.

Intracellular polymerizing compounds can be incorporated into a largerpeptide containing the targeting moiety and a linker. Suitable peptidesinclude the dipeptide ester referenced in Table 5 (i.e.,L-Leucyl-L-Leucine-O-Me). When transported into cells, these dipeptideesters preferentially accumulate in lysosomes and secondary granules ofcytotoxic cells. These dipeptides also undergo self-association andpolymerization, which results in trapping at low concentrations, andmembrane rupture at higher concentrations.

                  TABLE 6    ______________________________________    POLYMERIZING DI-PEPTIDE ESTER:    L-LEUCYL-L-LEUCINE-O--ME    ______________________________________    J. Invest. Dermat. 99:805-825, 1992    J. Clin. Invest. 84:1947-56, 1989    Transpl. 53:1334-40, 1992    J. Immunol. 138:51-7, 1987    J. Immunol. 148:3950-7, 1992    J. Immunol. 136:1038-48, 1986    Cryobiology 29:165-74, 1992    Acta. Biochem Biophys. Hung 24:299-311, 1989    Blood 79:964-71, 1992    Blood 78:2131-8, 1991    J. Immunol. 139:2137-42, 1987    J. Exp. Med. 172:183-194, 1990    J. Clin. Invest. 78:1415-20, 1986    PNAS 87:83-7, 1990    J. Immunol. 137:1399-406, 1986    PNAS 82:2468-72, 1985    ______________________________________

Suitable intracellular polymerizing compounds also include peptides thatcan self-associate into alpha-helical structures termed "leucinezippers". In the context of this invention, such structures may be usedto form intracellular polymers that are incapable of exitingintracellular vesicles. Such sequences can be selected by observing selfassociation of the compounds in solution, and the formation of polymerscapable of binding to DNA. Suitable peptide sequences that canself-associate into alpha helical structures are presented in Table 7.

                  TABLE 7    ______________________________________    LEUCINE ZIPPERS    ______________________________________    Boc(t-butoxycarbonyl)-Aib(alpha-aminoisobutyryl)    Glu(OB.sub.n l)-(benzoyl ester)-Leu--Aib--Ala--Leu--Aib--Ala--    Boc--Aib--Leu--Aib--Aib--Leu--Leu--Aib--Leu--Aib--O--Me    Proteins 12:324-30, 1992    Lys(Z)(benzyloxy-carbonyl)-Aib--O--Me    PNAS 87:7921-5, 1990    GELEELLKHLKELLKGER    Biochem. 31:1579-84, 1992    ______________________________________

In another embodiment of the present invention, a third functional classof rerouting moieties is disclosed. This class includes moieties thatcan be recognized by intracellular receptors. Such sequences areidentified by their ability to stop movement of endogenously synthesizedproteins to the cell surface. Suitable peptides include certain peptidesequences (such as sorting or signal sequences) associated with thetrafficking of endogenously synthesized proteins (Cur. Opin. Cell. Biol.3:634-41, 1991). Such peptide sequences, when covalently attached to theC-terminus of an exogenously added targeting moiety, result in theretention of the agent/receptor complexes in the endoplasmic reticulum("ER"), Golgi apparatus, or lysosomes.

Such peptide sequences are recognized by intracellular receptors,examples of which include both mammalian and bacterial versions of ERreceptors described in detail in J. Cell. Biol. 120:325-8, 1993; Embo.J. 11:4187-95, 1992; Nature 348:162-3, 1990. Further exemplary peptidesequences and variants thereof (shown in parentheses) that can berecognized by intracellular receptors are set forth in Table 8, SectionsA and B.

Certain signal sequences may be preferred for retention by one type oforganism versus another type. For example, REDLK is a preferred sequencerecognized by prokaryotic cells and to a lesser degree by eukaryoticcells (see Table 8, section C). Thus, employing this sequence as thererouting moiety, receptor modulating agents can be constructed toselectively inhibit a receptor-mediated process in bacteria, whilehaving little effect on mammalian cells.

                  TABLE 8    ______________________________________    PEPTIDE SEQUENCES WHICH BIND    INTRACELLULAR RECEPTORS    ______________________________________    A. Endoplasmic Reticulum or Golgi Retention Peptides    1. KDEL (DKEL,              J. Biol. Chem. 265:5952-5, 1990    RDEL, KNEL,              Biochem. Biophys. Res. Commun. 172:1384-91, 1990    SDEL, KEEL,              J. Virol. 65:3938-42, 1991    QDEL, KBDL,              Exp. Cell Res. 197:119-24, 1991    KDEL)     Growth Factors 5:243-53 1991              J. Biol. Chem. 267(10):7022-6, 1992              J. Biol. Chem. 267:10631-7, 1992              J. Cell. Biol. 118:795-811, 1992              J. Cell. Biol. 119:85-97, 1992              Exp. Cell. Res. 203:1-4, 1992              P.N.A.S. 90:2695-9, 1993              Mol. Biochem Parasitol 48:47-58, 1991              Embo J. 4:2345-55, 1992              J. Biol. Chem. 266:14277-82, 1991              Mol. Cell Biol. 11:4036-44, 1991    2. HDEL (HVEL,              J. Biol. Chem. 268:7728-32, 1993    HNEL, HTEL,              Mol. Biochem Parasitol 57:193-202, 1993    TEHT, DDEL,              J. Cell SCI 102:261-71, 1992    HIEL)     Eur J. Biochem. 206:801-6, 1992              J. Biol. Chem. 266:20498-503, 1991    3. ADEL   Embo J. 11:1583-91, 1992    4. REDLK  J. Biol. Chem. 266:17376-81, 1991    5. SEKDEL Growth Factors 5:243-53 1991    6. KTEL   J. Virol. 66:4951-6, 1992    B. Lysosomal Retention Peptides    1. KFERQ  Trends Biochem SCI 15:305-9 1990    2. Tyrosine-              J. Cell Biol. III:955-66, 1990    containing    polypeptides    C. ORGANISM-SPECIFIC RETENTION PEPTIDES    1. REDLK  J. Biol. Chem. 266:17376-17381, 1991    D. CLATHRIN-BINDING PEPTIDES (INTERNALIZATION SIGNALS)    1. LLAV   J. Cell. Biol. 199:249-57, 1992    2. YKYSKV J. Cell. Biol. 199:249-57, 1992              Embo. J. 7:3331-6, 1988    3. PPGYE  Cell 67:1203-9, 1991              Curr. Opin. Cell Biol. 3:1062, 1991    ______________________________________

A further class of peptide sequences of this invention, termed"internalization signals," function by binding to clathrin, both in thecoated pits, as well as those intracellular vesicles which maintain aclathrin coat. Representative examples of such clathrin-binding peptides(CBP) are disclosed in Table 8, section D. The CBP binds clathrin in thecoated pits initially located on the cell surface causing retention ofthe targeting moiety to which it is conjugated.

A further class of moieties capable of recognizing intracellularreceptors includes carbohydrates. Suitable carbohydrates include anycarbohydrate which is capable of binding to intracellular carbohydrate(CHO) receptors but not cell surface CHO receptors. Such carbohydratesinclude: mannose-6-phosphate and glucose-6-phosphate. Suitablecarbohydrate moieties include those which bind to the insulin-likegrowth factor II/mannose-6-phosphate (IGF II/M6P) receptor, includeanalogs of mannose-6-phosphate, as well as other phosphorylatedsaccharides (Carbohydrate Res. 213:37-46, 1991; FEBS Lett. 262:142-4,1990).

The affinity of the rerouting moiety can be varied by changes in thechemical nature of the phosphorylated saccharides (J. Biol. Chem.264:7970-5, 1989; J. Biol. Chem. 264:7962-9, 1989) (monosaccharides bindwith the lowest affinity, while di- or tri-saccharides bind withincreasingly higher affinity). Clustering of phosphorylated saccharideson protein carriers can dramatically increase affinity to theintracellular receptor.

Synthesis of various oligosaccharides are reviewed in Sem. Cell. Biol.2:319-326, 1991. Although, mannose-6-phosphate receptor expression isprimarily intracellular, expression also occurs on cell surfaces. Thus,in the context of the present invention, covalent attachment of atargeting moiety with a carbohydrate which binds the mannose-6-phosphatereceptor should be constructed so as to give at least 100-folddifference in binding affinity between the targeting moiety and thererouting moiety. For example, a vitamin B₁₂ /transcobalamin II receptortargeting moiety, in this case vitamin B₁₂, would have a bindingaffinity for the carrier protein, transcobalamin II (TcII), of ≧10⁻¹⁰ Mand an affinity for the IGF II/M-6-P receptor of 10⁻⁸ M or less. Thiswill maintain the specificity of the vitamin B₁₂ binding (via TcII),while allowing transfer of the receptor modulating agent from serumM-6-P soluble receptor to cell surface receptor.

In addition to IGF II/M-6-P receptor moieties, other carbohydrate-basedrerouting moieties also promote retention of the modulatingagent/receptor complex in the ER or Golgi complex. Such moieties arebased on the recognition by various glycosyl transferases ofcarbohydrate moieties, either as a natural substrate or as an inhibitor.Such moieties are reviewed in Sem. Cell. Biol. 2:289-308, 1991. Forexample, saccharide recognition moieties include penultimate sugars,such as glucose and N-acetyl glucosamine (which are natural substrates).More preferred, however, are glycosylation inhibitors which arerecognized by glycosyl transferases, but cannot serve to append furthercarbohydrate residues on growing chains (Sem. Cell. Biol. 2:309-318,1991) (see FIG. 7).

In yet another embodiment of the present invention, a fourth functionalclass of rerouting moieties is disclosed. This class is generallycomprised of rerouting moieties which anchor the receptor to the cellmembrane. By way of example, this class includes membrane-bindingpeptides that exhibit conditional pH-dependent membrane binding. Suchpeptides exhibit α-helical character in acid but not neutral pHsolutions. When a conditional membrane-binding peptide assumes a helicalconformation at an acidic pH, it acquires the property ofamphiphilicity, (e.g., it has both hydrophobic and hydrophilicinterfaces). More specifically, within a pH range of approximately5.0-5.5, such a peptide forms an alpha-helical, amphiphilic structurethat facilitates insertion of the peptide into a target membrane. Analpha helix-induced acidic pH environment may be found, for example, inthe low pH environment present within cellular endosomes or lysosomes.In aqueous solution at physiological pH, a conditional, membrane-bindingpeptide is unfolded (due to strong charge repulsion among charged aminoacid side chains) and is unable to interact with membranes.

Suitable conditional membrane-binding peptide sequences include thecharged amino acids glutamate, aspartate, and histidine. A preferredconditional membrane-binding peptide includes those with a highpercentage of helix-forming residues, such as glutamate, methionine,alanine, and leucine. Further, conditional membrane-binding peptidesequences include ionizable residues having pKas within the range of pH5-7, so that a sufficiently uncharged membrane-binding domain will bepresent within the peptide at pH 5 to allow insertion into the targetcell membrane. Conditional membrane-binding peptides can be incorporatedthrough covalent bonds to a chemical or peptide targeting moiety orsynthesized as an entire peptide sequence including a linker and peptidetargeting moiety.

A particularly preferred conditional membrane-binding peptide isaa1-aa2-aa3-EAALA(EALA)₄ -EALEALAA-amide, which represents amodification of a published peptide sequence (Biochemistry 26:2964,1987). Within this peptide sequence, the first amino acid residue (aa1)is preferably a unique residue such as cysteine or lysine, thatfacilitates chemical conjugation of the conditional membrane-bindingpeptide to a targeting protein. The peptide can also be incorporatedinto a fusion protein with a protein or peptide targeting moiety (seeExample 7). Amino acid residues 2-3 (i.e., aa2-aa3) may be selected tomodulate the affinity of the translocating peptide for differentmembranes. For instance, if both residues 2 and 3 are lysine orarginine, the peptide will have the capacity to bind to membranes orpatches of lipids having a negative surface charge. If residues 2-3 areneutral amino acids, the peptide will insert into neutral membranes.

Yet another preferred conditional membrane-binding peptide can bederived from sequences of apo-lipoprotein A-1 and B; peptide toxins suchas melittin, bombolittin, delta hemolysin and the pardaxins; antibioticpeptides, such as alamethicin; peptide hormones, such as calcitonin,corticotrophin releasing factor, beta endorphin, glucagon, parathyroidhormone, and pancreatic polypeptide. Such peptides normally bindmembranes at physiologic pH but through attachment of substituents thepeptides can be enhanced in their ability to form alpha-helices atacidic pH and reduced in their membrane-binding at physiologic pH. Anexample of such a modified peptide having pH-dependent membrane bindingat acidic pH is fully succinylated melittin. In this example, a peptide(melittin) that normally binds to membranes at physiological pH isconverted to a pH-dependent peptide through succinylation of lysines.Upon succinylation, the peptide displays an amphipathic character onlyat acidic pHs.

Insertion of a conditional membrane-binding peptide into a target cellmembrane is enhanced through stabilization of the amphiphilic alphahelix. Helix stabilization may be achieved: (1) by adding repeating"EALA" units to form a longer peptide; (2) by placing an amide at theC-terminus of the peptide, in order to counteract the helical dipole;(3) by polymerizing the peptide; (4) by substituting a naturalhelix-former for one or more of the stacked glutamates; or (5) byattaching the peptide to a targeting moiety through use of a longerlinker, in order to provide sufficient distance between the membranebinding peptide and the targeting moiety for the peptide to contact andinteract with the target cell intracellular membranes.

In yet another embodiment of the present invention, a fifth functionalclass of rerouting moieties is disclosed. In this context, the reroutingmoiety merely functions as a modulating agent in that the moietydisables the receptors by crosslinking the same. This class includes bi-or multi-valent receptor crosslinking moieties formed from monovalentbinding targeting moieties. Cross-linking of receptors in some receptorsystems is sufficient to cause a rerouting of cell surface receptors tolysosomes for degradation, rather than their normal pathway of receptorrecycling. The synthesis of a bivalent receptor modulating agent isexemplified in greater detail in the examples below.

A preferred cross-linking receptor modulating agent is a vitamin B₁₂dimer. In this embodiment, each vitamin B₁₂ molecule acts as a targetingagent and a rerouting agent; cross-linking the B₁₂ dimer will cross-linkthe vitamin B₁₂ receptors, thus impeding the receptor traffickingpathway. A preferred vitamin B₁₂ dimer is generally comprised of twovitamin B₁₂ molecules, such as cyanocobalamin, coupled by one or morelinkers through coupling sites independently selected from a-g, h(ribose), and i (benzimidazole). Preferably, cross-linking occursbetween d- coupling sites on both molecules d- or e-. The dimer must becapable of forming a B₁₂ /TcII complex. As noted above, thischaracteristic may be assayed using any one of several techniques knownin the art, including competitive binding assays.

A vitamin B₁₂ may be coupled to a second vitamin B₁₂ molecule in thesame manner as described in detail for conjugation of rerouting moietiesto vitamin B₁₂ targeting moieties. As noted above, dimers may besynthesized using one or more linkers of various lengths and anycombination of homobifunctional, heterobifunctional, homotrifunctional,or heterotrifunctional linkers. As noted above, the use of atrifunctional linker allows for coupling with any number of additionalmoieties.

In selecting a linker for dimer synthesis, it should be noted that thetotal number of atoms comprising the linker between the vitamin B₁₂molecules should generally be greater than 10 atoms, typically be in therange of 30 to 55 atoms and, preferably be 45. As noted above, one ofordinary skill in the art will appreciate that although the number ofatoms is calculated relative to a linear chain of atoms, linear chain,branched chain, and cyclical chain linkers or combinations thereof wouldbe suitable. Hence, the structure of the atom chain in a linker wouldinclude, by way of example, alkyl, heteroalky, alkylaryl, andheteroalkyl aryl.

By way of example, a dimer may be synthesized by combining two differentvitamin B₁₂ linker adducts in the presence of a coupling agent. Thelinkers couple and dimers may then be separated and purified using thesame methods outlined above.

Alternatively, activated vitamin B₁₂ may simply be combined with ahomobifunctional or homotrifunctional linker (Tables 1 and 3).Preferably, in this embodiment, the ratio of vitamin B₁₂ to linkershould be in the range of 2:1. Preferably, a 1:1 ratio is used inpreparation of mixed dimers (e.g., b- and e-acid derivatives) or mixedligands (e.g., B₁₂ and hormone). Dimers may be separated and purified asnoted above.

In still another alternative, vitamin B₁₂ linker adducts, synthesized asdescribed, above may be coupled by a third linker. The third linker, a"cross-linker," serves to bridge the linkers on the vitamin B₁₂ linkeradducts. Suitable cross-linkers include those noted in Tables 1, 2, and3.

Polymerization of peptides may be accomplished by placing a cysteineresidue at each end of a peptide, followed by oxidation using dissolvedoxygen or other mild oxidizing agent, such as oxidized glutathione. Theaverage length of a polymerized peptide may be controlled by varying thepolymerization reaction conditions.

The amino acid sequence of any of the peptides of this invention may beselected to include all L-amino acids or all D-amino acids having a sidechain pKa from 5.0 to 9.0. D-amino acids may be advantageously used toform non-proteolyzable peptides, since the D-amino acids are notmetabolized within the cell. Further, the peptides of the presentinvention may include a combination of L- and D-amino acids, whereinD-amino acids are substituted for L-amino acids on either side of aproteolytic cleavage site. Yet another preferred noncleavable peptideincorporates peptide bond analogs that are not susceptible toproteolytic cleavage by cellular enzymes.

As discussed above, the receptor modulating agents of this inventioncomprise a targeting moiety coupled to the rerouting moiety. Thererouting moieties identified above may be covalently attached to thetargeting moiety by any one of several techniques known in the art,including (a) by chemical modifications such as a disulfide formation,thioether formation, amide formation or a reduced or non-reducedSchiff's base, (b) by direct peptide bond formation as in a fusionprotein, or (c) by use of a chemical and peptide linker. Suitablepeptide linkers in this regard correspond to two or more amino acidresidues that allow the rerouting peptide to assume its activeconformation independent of its interaction with the targeting moiety,and which allows sufficient distance for rerouting moiety access to, forexample, intracellular membranes from the peptide attachment site on thetargeting moiety.

In one embodiment, a rerouting moiety may be conjugated to a vitamin B₁₂targeting moiety by any one of several means, including, by way ofexample, coupling a rerouting moiety to a reactive group on a vitaminB₁₂ linker adduct; coupling a vitamin B₁₂ to a reactive group on arerouting moiety linker adduct or an appropriate side chain thereof;coupling a vitamin B₁₂ linker adduct to a rerouting moiety linker adductor an appropriate side chain thereof; coupling a rerouting moiety/biotinbinding protein conjugate to a vitamin B₁₂ /biotin conjugate; orcoupling a rerouting moiety biotin conjugate to a vitamin B₁₂ /biotinbinding protein conjugate.

Coupling of a rerouting moiety to a vitamin B₁₂ linker adduct, or avitamin B₁₂ to a rerouting moiety linker adduct, may be accomplishedusing the same techniques noted above for coupling a vitamin B₁₂molecule with a linker. The only critical consideration of this aspectof the invention is that the total linker length must be sufficient toavoid steric hindrance. Preferably, the total linker length is at least6 atoms.

Coupling of a rerouting moiety/biotin binding protein conjugate to avitamin B₁₂ /biotin conjugate may be accomplished using any one ofseveral means described in detail in Avidin-Biotin Chemistry: AHandbook, ed. D. Savage, Pierce Chemical Co., 1992. Briefly, a biotinbinding protein conjugate is prepared using a rerouting moiety or, as ina second embodiment, a vitamin B₁₂ molecule. Suitable biotin bindingproteins include avidin or streptavidin. In some circumstances, a linkermay be utilized to distance the molecules. For example, when coupling avitamin B₁₂ to an avidin, a linker of at least 6 atoms is preferred.

A biotin conjugate is prepared using a vitamin B₁₂ molecule or, as in asecond embodiment, a rerouting moiety. By way of example, a vitamin B₁₂molecule is combined with an NHS ester of biotin. Preferably, thevitamin B₁₂ molecule is a vitamin B₁₂ linker adduct as described above.Even more preferably, the vitamin B₁₂ molecule is a vitamin B₁₂ linkeradduct characterized by a 12 atom linear linker coupled to the d- or e-coupling site.

Once formulated, coupling between the biotin conjugates and biotinbinding protein conjugates is easily accomplished by combining thecomplementing conjugates, ie., a vitamin B₁₂ /biotin conjugate with arerouting moiety/avidin conjugate.

In another aspect of the present invention, a B₁₂ /biotin conjugate isutilized to couple a vitamin B₁₂ to any number of compounds throughbiotin binding protein conjugates. Using a vitamin B₁₂ /biotinconjugate, any compound which is capable of coupling a biotin bindingprotein may be coupled to a vitamin B₁₂ and thereby internalized intocells expressing the vitamin B₁₂ receptor. Such compounds include, inaddition to the rerouting moieties described in detail below, hormones,enzymes, antibodies or fragments thereof, markers, or therapeutics.Coupling any of these compounds to a biotin binding protein, such asavidin or streptavidin, may be accomplished using techniques describedin detail in Avidin-Biotin Chemistry: A Handbook, ed. D. Savage, PierceChemical Co., 1992.

In one aspect of this embodiment, a vitamin B₁₂ /biotin conjugate iscoupled to a therapeutic/avidin conjugate directed at neoplasticdisorders. Neoplastic disorder therapeutics which may be coupled to avitamin B₁₂ /biotin conjugate through avidin include doxorubicin,daunorubicin, etoposide, teniposide, vinblastine, vincristin,cyclophophamide, cisplatin and nucleoside antimetabolites such asarabinosylcytosine, arabinosyladenine and fludarabine.

In another aspect of this embodiment, a vitamin B₁₂ /biotin conjugate iscoupled to a marker conjugated with a biotin binding protein. Suitablemarkers include, by way of example, fluorescent molecules orradiolabeled molecules. This combination may be utilized as a detectionsystem incorporated into a screening device to identify patients withlow receptor bearing cells or in the evaluation of receptorup-regulation, for example, following treatment of patients for any oneof a wide variety of receptor modulation disorders.

In another aspect of this embodiment, a vitamin B₁₂ /biotin conjugate iscoupled to a radioisotope conjugated to a biotin binding protein.Suitable radioisotopes include, any high energy emitting radioisotopescapable of conjugating a biotin binding protein. This combination may beutilized as a targeted radiodiagnostic or radiotherapeutic.

In yet another aspect of this embodiment, a vitamin B₁₂ /biotinconjugate is used to immobilize vitamin B₁₂ to a solid matrix oravidin-coated substrate. By way of example, this would enable one toisolate TcII, TcII receptors, and evaluate coupling sites on the VitaminB₁₂.

The receptor modulating agents of this invention regulatereceptor-dependent biological responses through alterations in thereceptor trafficking pathway. As illustrated in FIG. 1, with specificreference to the receptor for vitamin B₁₂, cell surface receptors areoften associated with clathrin-coated pits. When bound by the receptormodulating agent of the present invention, the coated pits invaginate toform vesicles. The vesicles are then directed by the rerouting agent tolysosomes for receptor degradation or delivered to endosomes where thererouting agent securely binds or delays the agent/receptor complex.Thus, the receptor modulating agents can incapacitate the receptorsnormally undergoing recycling.

Newly synthesized receptors will eventually replace the internalizedreceptor on the cell surface. However, this process is far more timeconsuming than recycling--many cells require hours or days to achievemaximal receptor re-expression. Continued exposure of the cell to thereceptor modulating agents will exhaust the intracellular receptorpools. Thus, by modulating a plasma membrane receptor, re-expression ofthe receptor can be substantially delayed, thereby regulating abiological response associated with that receptor for a prolonged periodof time.

Biological activity of receptor modulating agents of the presentinvention may be ascertained in vitro by any one of several means knownin the art including, competition binding assays or cell proliferationstudies. These techniques are described in detail in LaboratoryTechniques in Biochemistry and Molecular Biology: An Introduction toRadioimmunoassay and Related Techniques, 3rd Edition, ed. Burdon and vanKnippenberg, Elsevier, 1987. Biological activity may be evaluated invivo using techniques described in detail in Shieh et al., J. Immunol.152(2):859-866, 1994 in which human tumor cell lines are injected intonude mice, followed by therapy with receptor modulating agents. Next,tumor cells are removed, single cell suspensions prepared and TcII cellsurface receptor density may be evaluated by flow cytometry andbiotinylated vitamin B₁₂ and avidin FITC.

The receptor modulating agent of the present invention may beadministered in a therapeutically effective amount to treat a variety ofdisorders characterized in which control of the disease process orsymptoms can be achieved by modulation of one or more receptor systemsand the associated biological responses. Such disorders includeneoplastic disorders, autoimmune diseases, rheumatic arthritis,cardiovascular disease, and neurodegenerative diseases.

Common to many non-neoplastic disease processes is a stage in which thedisease process itself, or its symptoms, can be halted or ameliorated bythe use of an anti-proliferative agent such as vitamin B₁₂ /TcIIreceptor modulating agents. These commonly recognized stages include asensitization or elicitation phase in which immune cells responsible forthe disease become turned on by antigen specific or non-specific means,followed by a proliferative phase in which the immune cells expand innumber, and finally a symptomatic phase in which the expanded immunecells create tissue damage directly or indirectly. Neoplastic disordersinclude, by way of example, leukemia, sarcoma, myeloma, carcinoma,neuroma, melanoma, cancers of the breast, lung, liver, brain, colon,cervix, prostrate, Hodgkin's disease, and non-Hodgkin's lymphoma.Because of this, anti-proliferative chemotherapeutic drugs are commonlyutilized in the treatment of many diseases other than cancer, but arelimited in use to life threatening situations due to their associatedtoxicity. Anti-proliferative agents, such as the ones of the presentinvention (with little of the direct toxicity of chemotherapeuticdrugs), may be used more widely. More specifically, the vitamin B₁₂receptor modulating agents of the present invention are not destructiveto plasma membrane processes (e.g., ion transport). In addition, theanti-proliferative activity is reversible by administration of vitaminB₁₂. Furthermore, the agents of this invention may not be mutagenic,teratogenic, or carcinogenic since they act at the level of the plasmamembrane, and not at the level of the nucleus, and DNA by intercalationor cross-linking (as many chemotherapeutic drugs act).

An understanding of the pharmaceutical applications for B₁₂ /TcIIreceptor modulating agents requires a knowledge of the cell typestargeted by such therapy. To this end, various pharmaceuticalapplications are disclosed in Table 9 below.

                  TABLE 9    ______________________________________    TARGET CELLS FOR VITAMIN B.sub.12    RECEPTOR MODULATING AGENTS              OTHER         POTENTIAL              PROLIFERATION PHARMA-    TARGET    ASSOCIATED    CEUTICAL    CELL      MARKERS       APPLICATIONS    ______________________________________    Activated T-Cell              IL-2 receptor Graft versus Host Disease              Transferrin Receptor                            Organ Transplants              Insulin Receptor                            Auto-Immune Diseases              Class II Histocompati-                            Asthma              bility Antigens                            Crohn's Disease    Tumor Cells              Tumor Assoc. Ags.                            Tumor Therapy              Ki67          (alone and in combination              Transferrin Receptor                            with chemotherapeutic drugs)    Bone Marrow              CD-34         Allogeneic Bone Marrow    Stem Cells              Transferrin Receptor                            Transplants              Class II Histocompati-                            Reduction in Toxicity of              bility Antigens                            Chemotherapy              IL-1, IL-3 Receptors    Proliferating              Thy 1.1       Inhibition of Adhesions,    Fibroblasts              Transferrin Receptor                            Scarring              Insulin & Insulin-like                            Scleroderma              Growth-Factor              Receptors              Fibroblast Growth-              Factor Receptor    Proliferating              EGF Receptor  Psoriasis    Epithelium or              Proto-Oncogenes    Epidermal    (Keratinocytes)    ______________________________________

Proliferating and activated T-cells can cause a wide variety of diseasesranging from the chronic inflammation of Crohn's disease to more acuteorgan graft rejection. In all of these diseases, the T-cell may serve acentral pathogenic role or a more accessory role. Anti-proliferativechemotherapeutic drugs serve to reduce symptomotology and in some caseslead to long-term remission. Similarly, proliferating fibroblasts andepithelial cells may give rise to diseases characterized by cellovergrowth. Vitamin B₁₂ receptor modulating agents may be used toreplace or used in combination with existing chemotherapeutic regimensin these diseases. An important aspect of the use of anti-proliferativevitamin B₁₂ receptor modulating agents in these diseases is not to applyit so aggressively or with improper timing such that normal healing(adhesions, scarring) or cell renewal (psoriasis) processes are alsoinhibited. As such, low doses of receptor modulating agents may be usedduring healing and higher doses once healing is completed.Alternatively, receptor modulating agents may not be administered at alluntil after healing is completed.

As previously mentioned, B₁₂ /TcII receptor modulating agents can beused to deprive neoplastic cells of vitamin B₁₂. It has already beenshown that sufficient deprivation leads to the death of rapidlyproliferating lymphoid neoplasms such as leukemia and lymphoma.Moreover, short term treatment to reduce cellular availability of thisnutrient, combined with existing chemotherapeutic agents, markedlyimproves therapeutic efficacy.

For solid tumors, vitamin B₁₂ depletion may induce cytostasis anddifferentiation as well as cell death. Thus, B₁₂ /TcII receptormodulating agents may be used to induce differentiation in hormonallyresponsive solid tumors. An increase in the number of cells expressing adifferentiated phenotype should translate into an increase in expressionof hormone receptors. The hormone receptor status of tumors, such asbreast and prostrate cancer, are directly correlated with their responseto hormonal therapy. Accordingly, B₁₂ /TcII receptor modulating agentscan be used to increase the number of receptor positive tumor cells orincrease receptor density in order to enhance efficacy of subsequenthormonal therapy.

Vitamin B₁₂ receptor modulating agents may affect both replicatingneoplastic and normal cells. However, bone marrow progenitorsdemonstrate differential sensitivity or response. Thus, B₁₂ receptormodulating agents can be used to modulate sensitivity of bone marrowprogenitors so as to enhance their resistance to the toxic effects ofchemotherapeutic agents. Such chemotherapeutic drugs act primarily onreplicating cells, with non-replicating cells being much less sensitive.Antibodies are well suited for this application since delivery is morereadily achieved to highly accessible marrow versus normal organs andsolid tumors. In addition, a B₁₂ /TcII anti-receptor antibody,possessing the ability to modulate receptor, could differentially effectlymphoid versus epithelial tissues. Decreasing the sensitivity ofprogenitors to toxic drugs would increase the bone marrow reserves andenhance subsequent response to colony stimulating factors, and enablehigher doses of chemotherapy or reduce the interval to reconstitution.It should also be recognized that such positive effects on bone marrowprogenitors, as a natural consequence of B₁₂ receptor therapy forcancer, is an additional mechanism by which the therapeutic index ofchemotherapeutic drugs other than 5-FU and methotrexate can be improved.

In a variety of autoimmune diseases, graft versus host disease, ectopicallergy, and organ transplantation, an initial `induction` phase, inwhich the patient becomes sensitized to self or allo-antigens, isfollowed by a "proliferative" phase in which forbidden or unregulatedclones of B- or T-cells are expanded. It has long been known thattreatment with anti-proliferative, chemotherapeutic drugs followinginduction can inhibit expansion of forbidden clones, inhibit progressionof disease, and restore a stable state of tolerance. An antibody, OKT-3,that controls the proliferation of allo-antigen-sensitized T-cells, hasbeen approved for management of acute allograft rejection. Anti-receptorantibodies of the present invention can be substituted for extremelytoxic chemotherapeutic drugs or highly immunogenic antibodies such asOKT-3 and achieve a similar state of tolerance without these associateddrawbacks.

Inflammation is an application for which antibodies are already beingutilized in clinical trials. The primary emphasis has been on inhibitingthe early manifestations of inflammation by inhibiting recruitment orbinding of inflammatory cells to vascular endothelium of injured tissue.It also well recognized that proliferation of cells at the site ofinflammation contributes to the pathology and tissue destruction of bothacute as well as chronic inflammation. To this end, anti-proliferative,chemotherapeutic drugs have been widely used to inhibit sequelae ofinflammation.

Methotrexate is one such drug commonly used to treat symptoms associatedwith rheumatoid arthritis. The drug acts to reduce both localized (e.g.,synovium) and generalized inflammation associated with diseaseprogression. Methotrexate acts synergistically with vitamin B₁₂depletion in therapy of leukemia. B₁₂ receptor modulating agents cantherefore be combined with methotrexate to enhance efficacy inrheumatoid arthritis. Other methotrexate applications include treatingdestructive inflammation associated with chronic heart disease andcolitis.

Surgery, radiation or chemotherapy to the abdomen is often complicatedby the development of tissue adhesions. These represent a considerableclinical problem because they lead to bowel blockage and requiresurgical intervention. Peritoneal adhesions arise as a result ofproliferation of the cells of the peritoneal membrane lining theabdomen. A non-toxic means of interfering with such proliferation couldlead to restoration of these normal cells to homeostatic controlmechanisms and thereby inhibition of adhesion formation. A similarprocess of benign proliferation and subsequent scarring is acomplication of retinal surgery. Direct instillation of a small moleculeanalog of an antibody receptor antagonist could prevent such disablingcomplications.

The term "treatment" as used within the context of the presentinvention, refers to reducing or alleviating symptoms in a subject,preventing symptoms from worsening or progressing, inhibition orelimination of the causative agent, or prevention of the infection ordisorder in a subject who is free therefrom. Thus, for example,treatment of infection includes destruction of the infecting agent,inhibition of or interference with its growth or maturation,neutralization of its pathological effects and the like. A disorder is"treated" by partially or wholly remedying the deficiency which causesthe deficiency or which makes it more severe.

The receptor modulating agents of the present invention are administeredin a therapeutically effective dose. A therapeutically effective dosemay be determined by in vitro experiment followed by in vivo studies.

Pharmaceutical compositions containing the receptor modulating agents inan admixture with a pharmaceutical carrier or diluent can be preparedaccording to conventional pharmaceutical compounding techniques. Thecarrier may take a wide variety of forms depending on the form ofpreparation desired for administration (e.g., intravenous, oral topical,aerosol, suppository, parenteral or spinal injection). Preferably,administration is via stereotactical injection.

The following examples are offered by way of illustration, notlimitation.

EXAMPLES

In summary, the examples which follow disclose the synthesis of severalreceptor modulating agents of this invention utilizing differentfunctional classes of rerouting moieties. More specifically, a series ofexamples are presented which employ vitamin B₁₂ as a targeting moiety ina receptor modulating agent.

All chemicals purchased from commercial sources were analytical grade orbetter and were used without further purification unless noted.Isophthaloyl dichloride was purchased from Lancaster Synthesis Inc.(Windham, N.H.). All other reagents were obtained from Aldrich ChemicalCo. (Milwaukee, Wis.). Solvents for HPLC analysis were obtained as HPLCgrade and were filtered (0.2 μm) prior to use. Ion exchangechromatography was conducted with 200-400 mesh strongly basic anion 2%cross-linking Dowex-1-chloride (Aldrich Chemical Co). Amberlite XAD-2nonionic polymeric adsorbent and octadecyl functionalized silica gel forcolumn chromatography were obtained from Aldrich Chemical Co.

¹ H NMR were obtained on Bruker AC-500 (500 MHz) instrument. Thechemical shifts are expressed as ppm (δ) using tetramethylsilane asinternal reference. IR data were obtained on a Perkin-Elmer 1420infrared spectrophotometer. UV data were obtained on a Perkin-ElmerLambda 2 UV/V is spectrophotometer. Mass spectral data were obtained ona VG 7070H mass spectrometer using fast atom bombardment (FAB).

HPLC separations of compounds were obtained on Hewlett-Packardquaternary 1050 gradient pumping system with a UV detector. Analysis ofthe HPLC data were obtained on a Hewlett-Packard HPLC Chemstationsoftware.

HPLC for Monomers: HPLC separations were conducted at a flow rate of 1mL/min. on a 5 mm, 4.6 250 mm NH₂ column (RAININ microsorb-MV aminocolumn) eluting with 58 mM pyridine acetate, pH 4.4 in H₂ O:THF (96:4)solution. Retention times were: 1=4.3 min; 2=6.5 min; 3=8.0 min; 4=8.8min; 5=10.9 min; 6=2.3 min; 7=2.3 min; 8=3.0 min; 9=2.9 min; 10=2.9 min;13=3.4 min. Reverse-phase HPLC chromatography was carried out using aHewlett-Packard Lichrospher 100 RP-18 (5 mm, 125×4 mm) C-18 column usinga gradient solvent system at a flow rate of 1 mL/min. Solvent A in thegradient was methanol. Solvent B was H₂ O. Starting from an 40% A, thegradient was increased to 100% A over 10 min. The gradient was thenbrought back to 40% A over a 5 min period. Retention times under theseconditions for biotin conjugates were: 17=7.1 min; 18=7.2 min; 19=6.9min; 20=6.4 min.

Preparative LC was conducted to separate the mixture of monocarboxylicacids using RAININ Rabbit-plus peristaltic pumping system with a DYNAMAX(model UV-1) UV-visible absorbance detector at a flow rate of 0.15mL/min. ID column (Alltech, 150 psi), (1000 mm×25 mm) packed withaminopropyl silica (40-63 mm) was used.

HPLC for Dimers: For dimers 36, 37, and 38 solvent A in the gradient wasmethanol. Solvent B was H₂ O. The gradient was held at the startingmixture of 70% A for 2 min, then the percentage of A was linearlyincreased to 100% over the next 10 min. The gradient was held at 100% Afor 20 min. Retention times under these conditions for dimers were:36=8.7 min; 37=9.0 min; 38=8.9 min. For dimers 58-60 and 64-66 Solvent Ain the gradient was methanol. Solvent B was aqueous 1% acetic acid. Thegradient was begun at 40% A and was held at that composition for 2 min,then the percentage of A was linearly increased to 100% over the next 10min. Retention times for the compounds examined under these conditionswere: 58=14.0 min; 59=14.1 min; 60=13.9 min; 64=8.7 min; 65=8.6 min;66=9.0 min.

Example 1 Preparation and Purification of CyanocobalaminMonocarboxylates: Modification on the Corrin Ring

This example serves to demonstrate the hydrolysis of b-, d- ande-propionamide sites on a vitamin B₁₂ molecule using dilute acid inpreparation for coupling of a linker to the sites. Importantly, thehydrolysis of the b-, d- and e-propionamides is selective over thehydrolysis of a-, c- and g-acetamides, or the f-amide in theheterocyclic chain connecting the benzimidazole. An optimal yield ofmonocarboxylate to di- and tri-carboxylate derivatives was obtained atroom temperature in 0.1N HCl over a 10 day period. The non-hydrolyzedvitamin B₁₂ and the di- and tri-carboxylates produced were readilyisolated from the desired monocarboxylates by preparative liquidchromatography.

Specifically, cyanocobalamin (1) (3.7 mmol, 5 g) was dissolved in 500 mLof 0.1N HCl and stirred at room temperature for 10 days under argonatmosphere. The solution was then neutralized with 6N NaOH and thecobamides were desalted by extraction into phenol and applied to a 200 g(60×4 cm, 200-400 mesh) Dowex Cl⁻ ×2 column (acetate form; prepared bywashing with saturated sodium acetate until it was free from Cl⁻, thenwashing with 200 mL water). The column was eluted with water to removeunreacted cyanocobalamin and then eluted with 0.04M sodium acetate (pH4.67).

The first fraction of the elution contained three monocarboxylic acids.These were desalted by extraction into 100 mL of 90% (w/w) phenol, twicewith 25 mL and once with 10 mL of phenol. Three volumes of ethyl ether(3×160 mL) and 1 volume of acetone (160 mL) were added to the combinedphenol extracts. Monocarboxylic acids were removed from the organicphase by extraction with water (2×100 mL). The combined aqueous phaseswere extracted twice with 20 mL of ether to remove residual phenol. Theaqueous solution of monocarboxylic acids was evaporated to dryness.Yield: 2.5 g (50%).

The mixture of three acids (0.350 g) was then applied to a 200 g (1000mm×25 mm) column of aminopropyl coated silica (40-63 mm) and was elutedwith 58 mM pyridine acetate pH 4.4 in H₂ O:THF (96:4); the elute wascollected with an automatic fraction collector. The first eluted acidwas found to be b-monocarboxylic acid (2), the second eluted acid wase-monocarboxylic acid (3) and the third eluted acid was d-monocarboxylicacid (4). The acid fractions were desalted by phenol extraction. Thesolids obtained were crystallized from aqueous acetone.

b-acid (2): yield 0.122 g (35%), mp 267°-270° C. with decomposition, ¹ HNMR (MeOH-d₄, δ) 0.43 (s, 3H, C-20 CH₃); 1.00 (m, 2H); 1.18 (s, 3H, C-46CH₃); 1.24 (d, 3H, Pr₃ CH₃); 1.36 (br s, 9H, C-47 CH₃, C-54 CH₃); 1.4(s, 3H, C-25 CH₃); 1.9 (d, 7H, C-36 CH₃, C-30 CH₂, C-48 CH₂); 2.26 (d,6H, B10 & B11, CH₃); 2.36 (d, 2H, C-26 CH₂); 2.57 (s, 10H, C-35 CH₃,C-31 CH₂, C-37 CH₂, C-53 CH₃); 2.8 (m, 2H, C-60 CH₂); 3.3 (m, 3H, C-8H,C-13H); 3.6 (m, 2H, Pr₁ CH₂); 3.7 (d, 1H, R₅); 3.9 (d, 1H, R₅); 4.0 (m,1H, R₄); 4.12 (d, 1H, C-19); 4.17 (s, 1H, C-3); 4.3 (m, 1H, R₂); 4.5 (m,1H); 4.7 (m, 1H, R₃); 6.0 (s, 1H, C-10); 6.2 (s,1H, R₁); 6.5 (s,1H, B4);7.1 (s, 1H, B2); 7.2 (s, 1H, B7). MS (FAB⁺): m/e 1357 (M⁺ +1). IR (KBr):3400, 3200, 2950, 2060, 1660, 1570, 1490, 1060 cm⁻¹. UV (MeOH): λ360(ε23441)

e-acid (3): yield 0.168 g (48%), mp 245°-250° C. with decomposition, ¹ HNMR (MeOH-d₄, δ) 0.43 (s, 3H, C-20 CH₃); 1.01 (m, 2H); 1.15 (s, 3H, C-46CH₃); 1.23 (d, 3H, Pr₃ CH₃); 1.36 (br s, 9H, C-47 CH₃, C-54 CH₃); 1.4(s, 3H, C-25 CH₃); 1.83 (s, 4H, C-55 CH₂); 1.93 (m, 6H, C-36 CH₃, C-30CH₂, C-48 CH₂); 2.22 (d, 6H, B10 & B11 CH₃); 2.35 (s, 3H,C-26 CH₂); 2.5(d, 13H, C-35 CH₃, C-31 CH₂, C-37 CH₂, C-53 CH₃); 2.9 (m, 1H, C-60 H);3.2 (m, 1H, C-13H); 3.4 (m, 1H, C-8 H); 3.6 (d, 1H, Pr1 CH); 3.7 (d,1H); 3.9 (d, 1H); 4.0 (m, 2H); 4.1 (d, 1H); 4.2 (m, 2H); 4.6 (m, 1H);6.0 (s, 1H, C-10); 6.3 (d, 1H, R1); 6.5 (s, 1H, B4); 7.0 (s, 1H, B2);7.2 (s, 1H, B7). MS (FAB⁺): m/e 1357 (M⁺ +1). IR (KBr): 3400, 3200,2950, 2060, 1660, 1570, 1490, 1060 cm⁻¹. UV (MeOH): λ360 (ε21 842)!

d-acid (4): yield 0.060 g (17%), mp>300° C., ¹ H NMR (MeOH-d₄, δ) 0.43(s, 3H, C-20 CH₃); 1.04 (m, 2H); 1.15 (s, 3H, C-46 CH₃); 1.25 (d, 3H,Pr₃ CH₃); 1.36 (br s, 9H, C-47 CH₃, C-54 CH₃); 1.4 (s, 3H, C-25 CH₃);1.85 (s, 4H); 2.01 (s, 6H); 2.23 (d, 8H, B10 & B11 CH₃); 2.38 (d, 3H,C-26 CH₂); 2.53 (d, 13H, C-36 CH₃, C-30 CH₂, C-48 CH₂); 2.6 (m, 5H); 2.9(m, 1H, C-60 H); 3.3 (d, 1H, C-13H); 3.4 (m, 1H, C-8 H); 3.6 (d, 1H, Pr₁CH); 3.7 (d, 1H); 3.9 (d, 1H); 4.0 (m, 2H); 4.1 (d, 1H); 4.3 (m, 2H);6.0 (s, 1H, C-10); 6.3 (d, 1H, R1); 6.5 (s, 1H, B4); 7.1 (s, 1H, B2);7.2 (s, 1H, B7); UV (eOH): λ360 (ε22 127). MS (FAB⁺): m/e 1357 (M⁺ +1).IR (KBr): 3400, 3200, 2950, 2060, 1660, 1570, 1490, 1060 cm⁻¹.

Example 2 Cyanocobalamin Modified on Ribose: Succinate Conjugate (5)

This example serves to demonstrate the activation of the ribose couplingsite coupling site h (see structure I) with succinic anhydide.Cyanocobalamin (1) (0.15 mmoL, 200 mg) was dissolved in 40 mL ofdimethylsulfoxide (DMSO) containing 8 g (80 mmoL) of succinic anhydrideand 6.4 mL of pyridine. After 14-16 h at room temperature, the excess ofsuccinic anhydride was destroyed by adding 500 mL of water and keepingthe pH of the reaction mixture at 6 with 10% KOH. KCN was then added ata final concentration of 0.01M and the pH of the solution was readjustedto 6 with 3N HCl. After 1 h the cyanocobalamin components were desaltedby phenol extraction and applied to a 100 g of Dowex Cl⁻ (60×2.5 cm)column (acetate form, 200-400 mesh). The cyanocobalamin was eluted withwater. Succinate conjugate (5) was eluted with NaOAc (0.04 M, pH 4.67)which yielded 180 mg (85%) after isolation. The O2',O5'-disuccinylderivative remained absorbed on the column under these conditions. mp208°-210° C. with decomposition.

¹ H NMR (D₂ O-d₄, δ): 0.43 (s, 3H, C-20 CH₃); 0.95 (m, 2H); 1.15 (s,3H); 1.2 (d, 3H); 1.35 (d, 7H); 1.4 (s, 3H); 1.8 (s, 3H); 1.9 (s, 12H);2.2 (d, 6H); 2.36 (d, 2H); 2.5 (d, 10H); 2.6-2.7 (m, 7H); 3.0 (m, 1H);3.3 (d, 1H); 3.37 (m, 1H); 3.5 (d, 1H); 4.0 (d. 1H); 4.18 (m, 2H); 4.25(m, 3H); 4.54 (d, 1H); 6.0 (s, 1H); 6.3 (d, 1H); 6.4 (s, 1H); 7.0 (s,1H); 7.2 (s, 1H). MS (FAB⁺): m/e 1455 (M⁺ +1). IR (KBr): 3400, 3200,2950, 2060, 1660, 1570, 1490, 1060 cm⁻¹ ; UV (MeOH): λ360 (ε26041).

Example 3 Coupling of Cyanocobalamin Monocarboxylic Acids with1,12-Diaminododecane: Reaction without Sodium Cyanide

This example serves to demonstrate the coupling of a linker to acyanocobalamin monocarboxylate. Coupling of the monocarboxylates (2, 3,4) with diaminododecane was first attempted usingN-ethyl-N'-dimethylamino-propyl-carbodiimide hydrochloride (EDC) in H₂ Oaccording to Yamada and Hogenkamp, J. Biol. Chem. 247, 6266-6270, 1972.However, the products obtained did not have a reactive amino group.Alteration of the reaction conditions by changing the reaction mixtureto DMF/H₂ O and adding NaCN/N-hydroxysuccinimide (see Example 4) to thereaction mixture gave the desired diaminododecane adducts.

A mixture of cyanocobalamin monocarboxylic acid (0.370 mmoL, 500 mg) and1,12-diaminododecane (3.6 g) in 100 mL H₂ O was adjusted to pH 6 with 1NHCl. The solution was then treated withN-ethyl-N'-dimethylamino-propyl-carbodiimide-hydrochloride (EDC) (726mg) and stirred at room temperature for 22 h. In 5 intervals of 6 to 14h, 650 mg of EDC was added to the reaction mixture. After a totalreaction time of 4 days (HPLC monitoring) the solution was evaporated todryness, the residue was digested with 100 mL of acetone and the solventwas decanted. The solid residue was dissolved in 50 mL of water andapplied to an 175 g Amberlite XAD-2 (60×4 cm) column. Contaminates werewashed from the column with 1 L water, then the crude product was elutedwith 500 mL of methanol. The solution was evaporated to dryness, theresidue was dissolved in 25 mL of water and was applied to a 100 g DowexCl⁻ (60×2.5 cm) column (acetate form, 200-400 mesh). The final productwas eluted using 250 mL of water, thereby leaving non-converted acidbound to the column, which was later eluted with 0.04 mol/L sodiumacetate buffer pH 4.67. The fraction containing the final product wasevaporated to dryness.

The mass spectral value obtained indicated that HCN was lost from thedesired product. Further, ¹ H NMR data suggested that some protons werebeing affected by the cobalt. Thus, this reaction was conducted withNaCN (Example 4) to drive the equilibrium towards retention of Co--CN.N-hydroxy succinimide was also added to facilitate the couplingreaction.

e-acid adduct (6): Yield: 222 mg (40%). mp 172°-174° C. withdecomposition. ¹ H NMR (MeOH-d₄, δ): 0.43 (m, 3H, C-20 CH₃); 1.06 (t,4H, C-46 CH₃); 1.16 (m, 5H); 1.2 (m, 5H); 1.33 (m, 7H); 1.43 (s, 3H);1.68 (m, 4H); 1.86 (m, 5H); 2.2 (m, 8H); 2.3 (m, 6H); 2.4 (m, 10H); 2.55(m, 10H); 2.8 (m, 4H); 3.1 (m, 6H); 3.3 (m, 5H); 3.6 (m, 2H); 3.7 (m,2H); 3.8 (m, 1H); 4.0 (m, 1H); 4.1 (m, 1H); 4.16 (m, 1H); 4.3 (m, 1H);4.48 (m, 1H); 4.6 (m, 1H); 6.0 (d 1H, C-10); 6.2 (m, 1H, R1); 6.5 (m,1H, B4); 7.1 (m, 1H, B2); 7.2 (m, 1H, B7). MS (FAB⁺): m/e 1512. IR(KBr): 3400, 3200, 2950, 1660, 1570, 1490, 1060 cm⁻¹. UV (MeOH): λ360(ε21 877).

d-acid adduct (7): yield: 225 mg (45%), mp 195°-198° C. withdecomposition. ¹ H NMR (MeOH-d₄, δ): 0.43 (m, 3H, C-20 CH₃); 1.09 (m,7H); 1.14 (m, 6H); 1.2 (m, 10H); 1.27 (m, 10H); 1.33 (m, 6H); 1.5 (m,3H); 1.77 (s, 3H); 2.2 (m, 8H); 2.26 (s, 2H); 2.5 (m, 10H); 2.7 (m, 5H);3.0 (m, 2H); 3.1 (m, 2H); 3.2 (m, 3H); 3.5 (m, 2H); 3.6 (m, 1H); 3.8 (m,1H); 3.9 (m, 1H); 4.0 (m, 1H); 4.1 (m, 1H); 4.2 (m, 1H); 4.4 (m, 1H);4.6 (m, 1H); 6.0 (d 1H, C-10); 6.1 (m, 1H, R₁); 6.4 (m, 1H, B4); 7.0 (m,1H, B2); 7.1 (m, 1H, B7); MS (FAB⁺): m/e 1512, IR (KBr): 3400, 3200,2950, 1660, 1570, 1490, 1060 cm⁻¹ ; UV (MeOH): λ360 (ε22 680).

Example 4 Coupling of Cyanocobalamin Monocarboxylic Acids with1,12-Diaminododecane: Reaction Containing Sodium Cyanide

Cyanocobalamin monocarboxylic acid (2, 3, 4) (0.370 mmoL, 500 mg) andN-hydroxysuccinimide (1.48 mmoL, 170 mg) were dissolved in a mixture ofDMF: H₂ O (1:1) (18.4 mL) and 363 mg of NaCN was added.1,12-Diaminododecane was dissolved in a mixture of DMF: H₂ O (1:1) (18.4mL) and the pH was adjusted to 6 with 1N HCl. The diaminododecanesolution was then added in one portion to the cyanocobalamin solution.EDC (285 mg) was added and the pH of the solution was readjusted to 5.5.The reaction mixture was then stirred overnight in the dark at roomtemperature. In 5 intervals of 6-14 h, 170 mg of N-hydroxysuccinimideand 285 mg of EDC were added to the solution, readjusting the pH value5.5 each time. After a total reaction time of 4 days (reaction followedby HPLC), the solution was evaporated to dryness. The residue wasdigested with 100 mL of acetone and the solvent was decanted. The solidresidue was dissolved in 50 mL of H₂ O and applied to an 200 g AmberliteXAD-2 (60×4 cm) column. The column was eluted with 1 L water to removeundesired materials, then the desired product was eluted with 500 mLmethanol. The solution was evaporated to dryness, the residue wasdissolved in 25 mL of water and was applied to a 100 g Dowex Cl⁻ (60×2.5cm) column (acetate form, 200-400 mesh). The desired product was elutedfrom the column with 250 mL water, leaving any non-reacted acid bound tothe column. This was followed by elution with 0.04 mol/L sodium acetatebuffer pH 4.7. The fractions containing the final product wereevaporated to dryness.

b-isomer (8): yield 410 mg (82%), mp 172°-174° C. with decomposition. ¹HNMR (MeOH-d₄, δ) 0.43 (s, 3H, C-20 CH₃); 1.18 (s, 4H); 1.3 (m, 13H);1.39 (m, 13H); 1.45 (s, 5H); 1.6 (m, 4H); 1.72 (m, 2H); 1.9 (s, 6H);2.25 (d, 6H, B10 & B11 CH₃); 2.35 (m, 5H); 2.56 (m, 5H); 2.8-3.0 (m,8H); 3.15 (m, 4H); 3.3 (m, 2H); 3.4 (m, 2H); 3.6 (m, 1H); 3.68 (m, 1H);3.75 (m, 1H); 3.9 (d, 1H); 4.07 (m, 1H); 4.12 (d, 1H); 4.2 (br s, 1H);4.3 (m, 1H); 4.47 (m, 1H); 4.7 (m, 1H); 6.0 (s, 1H, C-10); 6.2 (d,1H,R₁); 6.5 (s,1H, B4); 7.1 (s, 1H, B2); 7.2 (s, 1H, B7); MS (FAB⁺): m/e1539 (M⁺ +1). IR(KBr): 3400,3200,2950,2060, 1660,1570,1490, 1060cm⁻¹. UV(MeOH): λ360 (ε15409).

e-isomer (9): yield: 430 mg (86%), mp 175°-180° C. with decomposition, ¹H NMR (MeOH-d₄, δ) 0.43 (s, 3H, C-20 CH₃); 1.17 (s, 4H, C-46 CH₃); 1.22(d, 4H, Pr₃ CH₃); 1.29 (s, 24H); 1.36 (br s, 6H); 1.4 (s, 6H); 1.6 (m,3H); 1.87 (s, 8H); 2.05 (m, 2H); 2.25 (s, 6H, B10 & B11 CH₃); 2.36 (m,3H); 2.55 (d, 10H); 2.8 (s, 4H); 3.06 (t, 2H); 3.1 (m, 3H); 3.3 (s, 1H);3.34 (m, 1H); 3.4 (m, 1H); 3.58 (m, 1H); 3.65 (m, 1H); 3.75 (d, 1H); 3.9(d, 1H); 4.0 (m, 1H); 4.1 (d, 1H); 4.16 (m, 1H); 4.3 (m, 2H); 4.48 (m,2H); 4.6 (m, 1H); 6.0 (s, 1H, C-10); 6.3 (d, 1H, R1); 6.5 (s, 1H, B4);7.0 (s, 1H, B2); 7.2 (s, 1H, B7); MS (FAB⁺): m/e 1539 (M⁺ +1). IR (KBr):3400, 3200, 2950, 2060, 1660, 1570, 1490, 1060 cm⁻¹. UV (MeOH): λ360(ε16 720)

d-isomer (10): yield: 400 mg (80%), mp 174°-178° C. with decomposition,¹ H NMR (MeOH-d₄, δ) 0.43 (s, 3H, C-20 CH₃); 1.07 (m, 3H, C-46 CH₃); 1.2(d, 4H, Pr₃ CH₃); 1.27 (m, 15H); 1.35 (br s, 9H); 1.42 (s, 3H); 1.53 (m,2H); 1.6 (m, 4H); 1.86 (s, 4H); 2.25 (d, 6H, B10 & B11 CH₃); 2.5 (d,10H); 2.8 (s, 3H); 2.9 (m, 6H); 3.15 (m, 3H); 3.2 (m, 4H); 3.4 (m, 3H);3.6 (d, 1H); 3.75 (d, 1H); 3.96 (d, 1H); 4.08 (m, 2H); 4.19 (m, 1H); 4.3(m, 2H); 4.65 (m, 1H); 6.0 (s, 1H, C-10); 6.3 (d, 1H, R₁); 6.5 (s, 1H,B4); 7.1 (s, 1H, B2); 7.2 (s, 1H, B7); UV (MeOH): λ360 (ε17 665). MS(FAB⁺): m/e 1539 (M⁺ +1). IR (KBr): 3400, 3200, 2950, 2060, 1660, 1570,1490, 1060 cm⁻¹.

Example 5 Coupling of Cyanocobalamin Monocarboxylic Acids withGamma-aminobutyric Acid (Gaba)

This example serves to demonstrate the coupling of a gamma-aminobutyricacid (GABA) linker to a vitamin B₁₂ molecule. This reaction scheme isrepresented in FIG. 9.

Gamma-aminobutyric acid (GABA) tert-butyl ester (11) (1 mmol) andcyanocobalamin monocarboxylates (2, 3, 4) (0.1 mmol.) are mixed in 20niL H₂ O and sufficient 0.1N HCl is added to adjust to pH to 6.0.N-ethyl-N¹ -dimethylaminopropylcarbodiimide hydrochloride (EDC) (0.5mmol) is added to the solution. The reaction mixture is stirred at roomtemperature for 24 hours and then the mixture is dried under vacuum.This reaction mixture is treated with TFA to remove the tert-butylester. A cyanocobalamin-GABA adduct (12) was purified. Reverse-phaseHPLC chromatography is carried out as described above. Acyanocobalamin-GABA adduct (12) can be further activated with acarbodiimide and coupled to a moiety as described below.

Example 6 Cyanocobalamin Modified on Ribose: Succinate-DiaminododecaneConjugate (13)

Cyanocobalamin-Ribose-Succinate (5) (0.370 mmoL, 538 mg) andN-hydroxylsuccinimide (1.48 mmoL, 170 mg) were dissolved in a mixture ofDMF:H₂ O (1:1) (18.4 mL) and 363 mg of NaCN was added. This reactionscheme is represented in FIG. 11. 1,12-Diaminododecane was taken in amixture of DMF: H₂ O (1:1) (18.4 mL), pH was adjusted to 6 with 1N HCl.The diaminododecane solution was then added in a portion to thecyanocobalamin solution. EDC (285 mg) was added, the pH of the solutionwas readjusted to 5.5 and the reaction mix. was stirred overnight in thedark at room temperature. In 5 intervals of 6 to 14 h 170 mg ofN-hydroxysuccinimide and 285 mg of EDC was added to the solution,readjusting the pH 5.5 each time. After a total reaction time of 4 days(HPLC monitored) the solution was evaporated to dryness, the residue wasdigested with 100 mL of acetone and the solvent was decanted. The solidresidue was dissolved in 50 mL of H₂ O and applied to an 200 g AmberliteXAD-2 (60×4 cm) column. Contaminates were washed from the column with 1L water and then the crude product was eluted with 500 mL methanol. Thesolution was evaporated to dryness, the residue was dissolved in 25 mLof water and was applied to a 100 g Dowex Cl⁻ (60×2.5 cm) column(acetate form, 200-400 mesh). The final product was eluted using 250 mLwater, thereby leaving non-converted acid bound to the column, which waslater eluted with 0.04 mol/L sodium acetate buffer pH 4.7. The fractioncontaining the final product (13) was evaporated to dryness. Yield: 425mg (70%), mp 185°-187° C. with decomposition.

¹ H NMR (MeOH-d₄, δ): 0.43 (s, 3H, C-20 CH₃); 1.15 (s, 3H); 1.2 (d, 3H);1.3 (s, 27H); 1.4 (m, 3H); 1.55 (m, 6H); 1.85 (m, 12H); 2.2 (d, 6H); 2.3(d, 6H); 2.5 (d, 10H); 2.8 (m, 10H); 3.0 (t, 3H); 3.1 (t, 3H); 3.2 (s,6H); 3.3 (m, 4H); 3.58 (m, 2H); 3.6 (d, 1H); 4.1 (d. 1H); 4.2 (m, 2H);4.3 (m, 1H); 4.4 (d, 1H); 6.0 (s, 1H); 6.2 (d, 1H); 6.5 (s, 1H); 7.1 (s,1H); 7.2 (s, 1H). MS (FAB⁺): m/e 1638 (M⁺). IR (KBr): 3400, 3200, 2950,2060, 1660, 1570, 1490, 1060 cm⁻¹ ; UV (MeOH): λ360.

Example 7 Modification of Cyanocobalamin Monocarboxylic Acids Conjugatedwith 1,12-Diaminododecane: Reaction with Succinic Anhydride

This example serves to demonstrate modification of an amino terminuslinking moiety to a carboxylate terminus. Such a modification may benecessary for conjugating amino containing rerouting agents (e.g.,aminosugars) to cyanocobalamin derivatives containing a linker.

Cyanocobalamin carboxylic acid diaminododecane conjugate (8, 9, 10)(0.138 mmoL, 200 mg) was dissolved in 40 mL of dimethylsulfoxide (DMSO)containing 8 g (80 mmoL) of succinic anhydride and 6.4 mL of pyridine.After 14-16 h at room temperature, the excess of succinic anhydride wasdestroyed by adding 500 niL of water and keeping the pH of the reactionmixture at 6 with 10% KOH. KCN was then added at a final concentrationof 0.01M and the pH of the solution was readjusted to 6 with 3N HCl.After 1 h the cyanocobalamin components were desalted by phenolextraction. The residue was digested with 100 mL of acetone and thesolvent was decanted. It was dissolved in 40 mL of H₂ O. 1N NaOH (2 mL)was added to it and the reaction was stirred at room temperature for15-20 min. It was then neutralized with 1N HCl and the cyanocobalamincomponents (14, 15, 16) were desalted by phenol extraction. Yield: 80 mg(40%); mp 190°-198° C. with decomposition.

¹ H NMR (MeOH-d₄, δ): 0.43 (s, 3H, C-20 CH₃); 1.17 (s, 4H, C-46 CH₃);1.23 (d, 4H, Pr₃ CH₃); 1.29 (s, 24H); 1.36 (br s, 6H); 1.4 (s, 6H); 1.87(s, 4H); 2.05 (m, 2H); 2.25 (s, 6H, B10 & B11 CH₃); 2.35 (m, 3H); 2.4(m, 5H); 2.55 (d, 10H); 2.7 (s, 5H); 2.8 (m, 2H); 3.1 (m, 6H); 3.3 (s,6H); 3.4 (m, 1H); 3.65 (m, 2H); 3.75 (d, 1H); 3.9 (d, 1H); 4.0 (m, 1H);4.1 (d, 1H); 4.16 (m, 1H); 4.3 (m, 1H); 4.48 (m, 1H); 4.6 (m, 2H); 6.0(s, 1H, C-10); 6.3 (d, 1H, R₁); 6.5 (s, 1H, B4); 7.1 (s, 1H, B2); 7.2(s, 1H, B7). MS (FAB⁺): m/e 1639 (M⁺). IR (KBr): 3400, 3200, 2950, 2060,1660, 1570, 1490, 1060 cm⁻¹. UV (MeOH): λ360 (ε22 564).

Example 8 Cyanocobalamin Modified on Monocarboxylic Acid:Diaminododecane-Biotin Conjugates

This example serves to demonstrate coupling a vitamin B₁₂ derivative andbiotin. Biotin conjugates (17, 18, 19) were obtained by reaction ofactivated cyanocobalamin monocarboxylic acid diaminododecane (14), (15),and (16) with the NHS ester of biotin (Sigma Chemical Co.).

To a solution of cyanocobalamin monocarboxylic acid diaminododecaneconjugate (14, 15, 16) (300 mg, 0.195 mmoL) in DMF (35 mL), was addedtriethylamine (0.027 mL, 0.195 nimoL). N-Hydroxysuccinimidobiotin (100mg, 0.295 mmoL) was then added over a period of 10-15 min and evaporatedto dryness. The solid residue was dissolved in 20 mL of water andapplied to an 75 g of Dowex Cl⁻ (40×2 cm) (acetate form, 200-400 mesh)column. The product was eluted using 250 mL of water. It was thenevaporated to dryness, the residue was dissolved in a 10 mL ofmethanol-water (7:3 v/v) and the solution was applied to a reverse phaseC-18 column (500 mm×25 mm, Alltech, 150 psi) which was developed withthe same solvent. RAININ Rabbit-plus peristaltic pumping system was usedwith a DYNAMAX (model UV-1) UV visible absorbance detector. The eluatewas collected with an automatic fraction collector. The fractionscontaining the final product (HPLC monitored) were evaporated todryness.

b-isomer (17): yield 159 mg (53%), mp 210°-212° C. with decomposition, ¹H NMR (MeOH-d₄, δ): 0.43 (s, 3H, C-20 CH₃); 1.18 (s, 4H); 1.3 (m, 13H);1.39 (m, 13H); 1.45 (s, 5H); 1.6 (m, 4H); 1.72 (m, 2H); 1.9 (s, 6H); 2.2(d, 8H, B10 & B11 CH₃); 2.6 (d, 12H); 2.7 (m, 3H); 2.8-3.0 (m, 8H); 3.1(m, 3H); 3.2 (m, 2H); 3.4 (s, 1H); 3.6 (m, 2H); 3.68 (d, 1H); 3.75 (m,1H); 3.9 (d, 1H); 4.07 (m, 1H); 4.12 (d, 1H); 4.2 (s, 1H); 4.3 (m, 1H);4.47 (m, 1H); 4.7 (m, 1H); 6.0 (s, 1H, C-10); 6.2 (d,1 H, R1); 6.5(s,1H, B4); 7.1 (s, 1H, B2); 7.2 (s, 1H, B7); MS (FAB⁺): m/e 1764 (M⁺).IR (KBr): 3400, 3200, 2950, 2060, 1660, 1570, 1490, 1060 cm⁻¹. UV(MeOH): λ360 (ε23 746).

Anal. Calcd. for C₈₅ H₁₂₇ N₁₇ O₁₆ CoPS·11H₂ O: C, 51.98; H, 7.59; N,12.13. Found: C, 51.91; H, 7.81; N, 12.31.

e-isomer (18): yield 174 mg (58%), mp 222°-224° C. with decomposition, ¹H NMR (MeOH-d₄, δ): 0.43 (s, 3H, C-20 CH₃); 1.17 (s, 4H, C-46 CH₃); 1.22(d, 4H, Pr₃ CH₃); 1.29 (s, 24H); 1.36 (br s, 6H); 1.4 (s, 6H); 1.6 (m,4H); 1.72 (m, 2H); 1.87 (s, 4H); 2.17 (m, 3H); 2.25 (s, 6H, B10 & B11CH₃); 2.36 (m, 3H); 2.55 (d, 10H); 2.64 (m, 2H); 2.8 (s, 4H); 2.97 (s,4H); 3.1 (m, 3H); 3.3 (m, 1H); 3.4 (m, 1H); 3.58 (m, 1H); 3.65 (m, 1H);3.75 (d, 1H); 3.9 (d, 1H); 4.0 (m, 1H); 4.1 (d, 1H); 4.16 (m, 1H); 4.3(m, 2H); 4.48 (m, 2H); 4.6 (m, 1H); 6.0 (s, 1H, C-10); 6.3 (d, 1H, R1);6.5 (s, 1H, B4); 7.0 (s, 1H, B2); 7.2 (s, 1H, B7); MS (FAB⁺): m/e 1764(M⁺). IR (KBr): 3400, 3200, 2950, 2060, 1660, 1570, 1490, 1060 cm⁻¹. UV(MeOH): λ360 (ε24 441).

Anal. Calcd. for C₈₅ H₁₂₇ N₁₇ O₁₆ CoPS·9H₂ O (13): C, 52.96; H, 7.53; N,12.35. Found: C, 52.85; H, 7.55; N, 12.30.

d-isomer (19): yield 165 mg (55%), mp 216°-218° C. with decomposition, ¹H NMR (MeOH-d₄, δ): 0.43 (s, 3H, C-20 CH₃); 1.16 (s, 3H, C-46 CH₃); 1.2(d, 4H, Pr₃ CH₃); 1.28 (s, 15H); 1.35 (br s, 9H); 1.42 (s, 3H); 1.53 (m,2H); 1.6 (m, 4H); 1.72 (m, 2H); 1.86 (s, 6H); 2.16 (m, 3H); 2.02 (m,4H); 2.25 (d, 6H, B10 & B11 CH₃); 2.5 (d, 10H); 2.7(d, 1H); 2.8 (m, 5H);3.1 (m, 6H); 3.2(m,3H); 3.4(m, 1H); 3.57 (m, 1H); 3.6 (d, 1H); 3.7 (d,1H); 3.9 (d, 1H); 4.0 (m, 1H); 4.11 (d, 1H); 4.17 (m, 1H); 4.3 (m, 2H);4.4 (m, 2H); 4.6 (m, 1H); 6.0 (s, 1H, C-10); 6.3 (d, 1H, R1); 6.5 (s,1H, B4); 7.1 (s, 1H, B2); 7.2 (s, 1H, B7); MS (FAB⁺): m/e 1764 (M⁺); IR(KBr): 3400, 3200, 2950, 2060, 1660, 1570, 1490, 1060 cm⁻¹ ; UV (MeOH):λ360 (ε29 824).

Anal. Calcd for C₈₅ H₁₂₇ N₁₇ O₁₆ CoPS·10H₂ O: C, 52.46; H, 7.56; N,12.24. Found: C, 52.27; H, 7.56; N, 12.34.

Example 9 Cyanocobalamin Modified on Ribose:Succinate-Diaminododecane-Biotin Conjugate (20)

This example serves to demonstrate the conjugation of the ribose-linkeddiaminododecane adduct (13) with biotin to produce a cyanocobalaminbiotin conjugate (20).

To a solution of (11) (300 mg, 0.183 mmoL) in DMF (35 mL), triethylamine(0.025 mL, 0.183 mmoL) was added. N-hydroxysuccinimidobiotin (100 mg,0.295 mmoL) was added over a period of 10-15 min. and then evaporated todryness. The solid residue was dissolved in 20 mL of water and adjustedto pH 10 with 1N NaOH and applied to an 75 g Dowex Cl⁻ (40×2 cm)(200-400 mesh) column. The water fraction was discarded. The product wasthen eluted with 0.1N NH₄ OAc and was desalted by phenol extraction. Theresidue was dissolved in a 10 mL of methanol-water (7:3 v/v) and thesolution was applied to a reverse phase column (octadecyl) which wasdeveloped with the same solvent. The fractions containing the finalproduct (20) (HPLC monitored) were evaporated to dryness. Yield 135 mg(45%), mp 198°-205° C. with decomposition.

¹ H NMR (MeOH-d₄, δ): 0.43 (s, 3H, C-20 CH₃); 1.15 (s, 3H); 1.2 (d, 3H);1.3 (s, 27H); 1.36 (m, 6H); 1.4 (m, 3H); 1.6 (m, 4H); 1.7 (m, 2H); 1.85(m, 12H); 2.0 (d, 3H); 2.17 (m, 3H); 2.2 (d, 6H); 2.3 (d, 6H); 2.5 (d,10H); 2.64 (m, 2H); 2.8 (m, 10H); 3.1 (m, 6H); 3.25 (m, 6H); 3.58 (m,2H); 4.0 (m, 1H); 4.1 (m, 1H); 4.16 (m, 1H); 4.4 (m, 1H); 4.6 (s, 2H);4.7 (m, 1H); 6.0 (s, 1H); 6.2 (d, 1H); 6.5 (s, 1H); 7.1 (s, 1H); 7.2 (s,1H). MS (FAB⁺): m/e 1866 (M⁺). IR (KBr): 3400, 3200, 2950, 2060, 1660,1570, 1490, 1060 cm⁻¹. UV (MeOH): λ360 (ε28 434).

Example 10 Synthesis of a Cyanocobalamin/Lysosomotropic Compound(Streptomycin) Receptor Modulating Agent

This example demonstrates coupling of streptomycin to a cyanocobalaminor cobalamin derivative. Streptomycin (21) is conjugated withcyanocobalamin monocarboxylate (2, 3, 4) or a diaminoalkylsuccinatederivative (14, 15, 16) through the use of an oxime coupled linkingmoiety (FIG. 13). The linking group, ((3-aminopropyl)aminoxy)acetamide(22) is prepared by reaction of the N-hydroxysuccinimidyl ester of1,1-dimethylethoxycarbonyl-aminooxyacetic acid (23) (J. Med. Chem.36:1255-126, 1993) with an excess of diaminopropane in anhydrous THF.The linking group is separated from other compounds in the reactionmixture by preparative chromatography. The linker (1 g) is then mixedwith streptomycin (0.5 g) in 10 mL of H₂ O containing sodium acetate.The aqueous solution is warmed in a H₂ O bath for 10 minutes to yield acrude streptomycin-linker adduct (25) which may be purified bychromatography on acid washed alumina (J. Am. Chem. Soc. 68:1460, 1946).The aqueous solution containing the streptomycin linker adduct (0.15mmol) is mixed with an aqueous solution of activated cyanocobalamin (2,3, 4) (01. mmol) and EDC (0.5 mmol) is added. The reaction mixture isstirred at room temperature for 24 hours, then run over a reversed-phasepreparative chromatography column for purification of thecyanocobalamin-streptomycin receptor modulating agent (26).

Example 11 Synthesis of a Cyanocobalamin/Lysosomotropic Compound(Acridine) Receptor Modulating Agent

This example demonstrates the coupling of the vitamin B₁₂ to acridine.Chloroquine, quinacrine and acridine are lysosomotropic dyes which arerelatively non-toxic and concentrated as much as several hundred fold inlysosomes. Acridine derivatives may be covalently attached to atargeting moiety (such as cyanocobalamin) by the reaction schemeillustrated in FIG. 14, method A, or similarly as described in method B.Both reaction schemes produce a cyanocobalamin-acridine conjugate.

Method A: A diamine side chain is first synthesized in a manneranalogous to the side chain of quinacrine. Specifically, mono-phthaloylprotected 1,4-diaminobutane (27) is reacted with6,9-dichloro-2-methoxyacridine (28) in phenol (J. Am. Chem. Soc.66:1921-1924, 1944). The reaction mixture is then poured into an excessof 2N NaOH and extracted with ether. The ether extract is washed with 1MNaHCO₃, then H₂ O, and dried over MgSO₄. The crude product isrecrystallized from H₂ O-alcohol. The phthaloyl protecting group isremoved using anhydrous hydrazine in MeOH (Bioconjugate Chem. 2:435-440,1991) to yield the aminoacridine, (29). Aminoacridine (29) is thenconjugated with vitamin B₁₂ monocarboxylic acid (2, 3, 4) to yield acyanocobalamin-acridine conjugate (30).

Method B: Acridine derivative (31) (0.098 mmol, 0.045 g) was dissolvedin 0.5 mL of trifluoroacetic acid. This solution was stirred at roomtemperature for 0.5 h. TFA was removed by aspirator vacuum. The residuewas dissolved in 5 mL of acetonitrile and was neutralized by few dropsof triethylamine. Acetonitrile was then removed by aspirator vacuum. Theresidue was dissolved in DMSO (10 mL) and cyanocobalamin carboxylicacid-diaminododecane-succinyl derivative (15, 16, 17) (0.098 mmol, 134mg) was added followed by triethylamine (12 μL). The reaction mixturewas then stirred at room temperature for 24 h. (HPLC monitored), andevaporated to dryness. The residue was digested with 100 mL of acetoneand the solvent was decanted yielding a cyanocobalamin-acridineconjugate (32). Yield: 120 mg (62%). mp 182°-188 ° C.

¹ H NMR (MeOH-d₄, δ): 0.43 (s, 3H, C-20 CH₃); 1.17 (s, 4H, C-46 CH₃);1.23 (d, 4H, Pr₃ CH₃); 1.29 (s, 24H); 1.36 (br s, 6H); 1.4 (s, 6H); 1.65(m, 2H); 1.87 (s, 4H); 2.05 (m, 2H); 2.25 (s, 6H, B10 & B11 CH₃); 2.35(m, 3H); 2.4 (d, 5H); 2.44 (d, 2H); 2.55 (d, 10H); 2.64 (s, 5H); 2.8-2.9(m, 8H); 3.1-3.15 (m, 6H); 3.3 (s, 6H); 3.4 (m, 1H); 3.65 (m, 2H); 3.75(d, 1H); 3.9 (d, 1H); 3.98 (s, 2H); 4.0 (m, 2H); 4.1 (d, 1H); 4.16 (m,1H); 4.3 (m, 1H); 4.48 (m, 1H); 4.6 (m, 2H); 6.0 (s, 1H, C-10); 6.3 (d,1H, R₁); 6.5 (s, 1H, B4); 7.1 (s, 1H, B2); 7.2 (s, 1H, B7); 7.3 (t, 1H);7.4 (dd, 1H); 7.6 (dd, 1H); 7.7 (2dd, 2H); 7.8 (d, 1H); 7.9 (d, 1H); 8.4(d, 1H).

Example 12 Synthesis of a Cyanocobalamin/Lysosomotropic Compound(Amikacin) Receptor Modulating Agent

This example demonstrates conjugation of amikacin to a cyanocobalaminmolecule to form a cyanocobalamin-amikacin conjugate. A reaction schemefor the conjugation is depicted in FIG. 12. As noted above, chemicalmoieties that are retained subcellularly within lysosomes are termedlysosomotropic. Aminoglycosides are lysosomotropic compounds, and thusmay be used as rerouting moieties of this invention. The primary longchain amine on the hydroxyaminobutyric acid side chain of theaminoglycoside, amikacin (see FIG. 3), is preferentially reactive.Specifically, amikacin (33) (Sigma Chemical Co., St. Louis), is reactedwith a vitamin B₁₂ monocarboxylate (2, 3, 4) in the presence of EDC. Acyanocobalamin-amikacin conjugate (34) is then separated and purified byreverse-phase LC chromatography under conditions noted above.

Example 13 Cyanocobalamin Monocarboxylic Acid Diaminododecane ConjugateDimer: Isophthaloyl Dichloride Cross-Linking

This example demonstrates the production of a cyanocobalamin dimersuitable for use as a cross-linking receptor modulating agent.Cross-linking of receptors in some receptor systems is sufficient tocause a rerouting of cell surface receptors to lysosomes fordegradation, rather than their normal pathway of receptor recycling.

To a solution of cyanocobalamin monocarboxylic acid diaminododecaneconjugate (8, 9, 10) (0.192 mmol, 0.300 g) in DMF (30 mL), was addedtriethylamine (18 μL). Isophthaloyl dichloride (35) (0.096 mmol, 0.0195g) was added over a period of 10-15 min. The reaction mixture wasstirred at 55°-60° C. for 48 h (HPLC monitored) and evaporated todryness. The solid residue was dissolved in 20 mL of methanol: H₂ O(7:3) and applied to a reverse phase C-18 column (500 mm×25 mm, Alltech,150 psi) which was developed with the same solvent. RAININ Rabbit-plusperistaltic pumping system was used with a DYNAMAX (model UV-1) UVvisible absorbance detector; the elute was collected with an automaticfraction collector. The fractions containing the final product (HPLCmonitored) were evaporated to dryness.

b-acid dimer (36): yield 96 mg (30%), mp 217° 220° C. withdecomposition, ¹ H NMR (D₂ O, δ) 0.43 (s, 6H, C-20 CH₃); 1.18 (s, 8H);1.3 (m, 36H); 1.37 (m, 12H); 1.46 (s, 10H); 1.6 (m, 8H); 1.9 (d, 12H);2.05 (m, 10H); 2.2 (d, 16H, B10 & B11 CH₃); 2.35 (m, 8H); 2.6 (d, 18H);2.8-3.0 (m, 16H); 3.15 (m, 6H); 3.3 (s, 8H); 3.37 (m, 14H); 3.6 (m, 4H);3.76 (m, 2H); 3.9 (d, 2H); 4.07 (m, 2H); 4.12 (m, 2H); 4.18 (m, 2H); 4.3(m, 2H); 4.5 (m, 2H); 4.6 (s, 2H); 4.68 (m, 2H); 6.0 (s, 2H, 2C-10);6.26 (d,2H, 2R1); 6.6 (s,2H, 2B4); 7.1 (s, 2H, 2B2); 7.25 (s, 2H, 2B7);7.54 (t, 1H); 7.95 (d, 2H); 8.25 (s, 1H); MS (FAB⁺): m/e 3208. IR (KBr):3400, 3200, 2950, 2060, 1660, 1570, 1490, 1060 cm⁻¹ ; UV: λ360 (ε42380).

e-acid dimer (37): yield 121 mg (38%), mp 220°-222° C. withdecomposition, ¹ H NMR (D₂ O, δ) 0.43 (s, 6H, C-20 CH₃); 1.17 (s, 8H);1.22 (d, 13H); 1.29 (s, 45H); 1.36 (d, 22H); 1.44 (s, 10H); 1.6 (m, 8H);1.87 (s, 8H); 2.04 (m, 10H); 2.25 (s, 12H, B10 & B11 CH₃); 2.36 (m, 8H);2.55 (d, 20H); 2.8 (m, 8H); 3.15 (m, 8H); 3.29 (s, 10H); 3.36 (m, 14H);3.6 (m, 4H); 3.73 (m, 2H); 3.9 (d, 2H); 4.07 (m, 2H); 4.12 (m, 2H); 4.16(m, 2H); 4.3 (m, 2H); 4.5 (m, 2H); 4.6 (s, 2H); 4.66 (m, 2H); 6.0 (s,2H, 2C-10); 6.26 (d,2H, 2R1); 6.6 (s,2H, 2B4); 7.1 (s, 2H, 2B2); 7.25(s, 2H, 2B7); 7.54 (t, 1H); 7.93 (d, 2H); 8.25 (s, 1H); MS (FAB⁺): m/e3208. IR (KBr): 3400, 3200, 2950,2060, 1660, 1570, 1490, 1060 cm⁻¹. UV(MeOH): λ360 (ε33 854)

d-acid dimer (38): yield 96 mg (30%), mp 225°-228° C. withdecomposition, ¹ H NMR (D₂ O, δ) 0.43 (s, 6H, C-20 CH₃); 1.16 (s, 8H);1.29 (m, 36H); 1.35 (d, 12H); 1.44 (s, 10H); 1.53 (m, 6H); 1.6 (m, 8H);1.85 (s, 12H); 2.03 (m, 8H); 2.25 (d, 12H, B10 & B11 CH₃); 2.33 (m, 8H);2.54 (d, 20H); 2.8 (m, 8H); 3.13 (m, 8H); 3.28 (s, 12H); 3.35 (m, 12H);3.6 (m, 4H); 3.73 (m, 2H); 3.9 (d, 2H); 4.07 (m, 2H); 4.12 (m, 2H); 4.16(m, 2H); 4.3 (m, 2H); 4.5 (m, 2H); 4.64 (m, 2H); 4.7 (s, 2H); 6.0 (s,2H, 2C-10); 6.26 (d,2H, 2R1); 6.6 (s,2H, 2B4); 7.1 (s, 2H, 2B2); 7.25(s, 2H, 2B7); 7.54 (t, 1H); 7.93 (d, 2H); 8.25 (s, 1H); MS (FAB+): m/e3208. IR (KBr): 3400,3200,2950,2060,1660,1570,1490,1060 cm⁻¹ UV(MeOH):λ360 (ε31 747).

Example 14 Cyanocobalamin Monocarboxylic Acid Diaminododecane ConjugateDimer: ETAC Cross-Linking

This example serves to illustrate synthesis of a bivalent receptormodulating agent using a heterotrifunctional cross-linker. The reactionscheme for this synthesis is depicted in FIG. 15. Theheterotrifunctional cross-linker is formed an ETAC reagent (BioconjugateChem. 1:36-50, 1990; Bioconjugate Chem. 1:51-59, 1990; J. Am. Chem. Soc.101:3097-3110, 1979). Bivalency, in addition to enhancing affinity ofbinding, also imparts the ability to cross-link neighboring receptorsand trigger endocytosis. The bivalent "arms" of the agent may belengthened with peptide or other linking molecules to enablesimultaneous binding of both "arms". In the case of vitamin B₁₂ this maybe assessed by gel filtration. If the linkers allow simultaneousinteraction, there will be 2 moles of TcII for every mole of ETAC dimerpresent in a single peak of 80,000 m.w. (versus 40,000 m.w. of monomericTcII). Simultaneous binding of 2 moles of TcII will then have thepotential for bivalent binding to cell surface receptor. This can betested by comparing the affinity of monomer and dimer binding toreceptor. While the bivalent agent can be synthesized to include anyrerouting moiety of this invention which enhances lysosomal targetingand retention, the compound tyramine, useful for radio-labeling isdisclosed for the purpose of illustration.

Referring to FIG. 15, carboxy-ETAC (39) is prepared by the method ofLiberatore et al. (Bioconjugate Chem. 1: 1990). The carboxy-ETAC isconverted to its acid chloride by reaction in thionyl chloride. Additionof amine (40) gives the amine-ETAC adduct (41). Reaction of amine-ETAC(1 mmol) in CH₃ CN with 1M aqueous cysteamine (10 mmol) is conducted bystirring at room temperature for 24 h. This compound is reduced withNaCNBH₃ under acidic conditions. The crude amine-ETAC-cysteamine adduct(42) is purified by reverse-phase LC, using conditions noted above. Avitamin B₁₂ monocarboxylate (2, 3, 4) is conjugated withtyramine-ETAC-cysteamine compound by reaction with EDC in H₂ O. Theresultant vitamin B₁₂ -ETAC-tyramine dimer (43) is purified by reversephase LC, using conditions described above.

Example 15 Cyanocobalamin Monocarboxylic Acid Diaminododecane ConjugateDimer: Isophthlate Cross-Linking with Biotin Moiety

This example illustrates the synthesis of a bivalent receptor modulatingagent which is additionally coupled to a biotin moiety (44). Furthermodification can be obtained by coupling of this molecule with an avidinor streptavidin moiety.

Reaction Step A: Biotin (12.3 mmol, 3 g) was dissolved in warm (bathtemperature 70° C.) DMF (60 mL) under argon atmosphere. It was then coolto ambient temperature and DCC (13.5 mmol, 2.79 g) was added, followedby tetrafluorophenol (24.6 mmol, 4.08 g). The reaction mixture was thencooled to 0° C. and stirred for 0.5 h. It was then brought back toambient temperature and stirred for another 4-5 h. The reaction mixturewas filtered and the filtrate was evaporated to dryness. The precipitatewas washed with acetonitrile (50 mL) and was filtered to yield 5 g (98%)of white solid (45).

¹ H NMR (DMSO, δ): 1.4 (m, 2H); 1.7 (m, 2H); 2.5 (t, 2H); 2.8 (t, 2H);3.1 (m, 1H); 4.1 (m, 1H); 4.3 (m, 1H); 6.4 (d, 2H); 7.9 (m, 1H).

Reaction Step B: 6-Aminocaproic acid (46) (7.5 mmol, 0.99g) wasdissolved in H₂ O (75 mL). Triethylamine (0.5 mL) was added followed bya solution of TFP ester of Biotin (5 mmol, 1.96 g) in warm acetonitrile(300 mL). The reaction was stirred overnight at room temperature. It wasthen filtered, washed with H₂ O (50 mL) and dried on high vacuum. Yield:0.870 g (47%). The filtrate was evaporated to dryness. The residue wastaken in boiling acetonitrile (75 mL) and was filtered, washed with hotacetonitrile. The solid (47) was dried on high vacuum to give 0.6 g, fora total yield of 1.47 g (79%).

¹ H NMR (DMSO-d₆, δ): 1.2-1.6 (m, 8H); 2.0 (t, 2H); 2.2 (t, 2H); 2.5(dd, 2H); 2.8 (dd, 2H); 3.1 (m, 3H); 4.1 (m, 1H); 4.3 (m, 1H); 6.4 (d,2H); 7.7 (m, 1H).

Reaction Step C: Biotin conjugated caproic acid (47) (2.68 mmol, 1 g)was dissolved in DMSO (50 mL). Triethylamine (0.4 mL) was added followedby TFP acetate (4.02 mmol, 1.05 g). The reaction mixture was thenstirred at room temperature for 15-20 min (HPLC monitored). It was thenevaporated to dryness. The residue was washed with ether anddichloromethane and dried on high vacuum (48). Yield: 1.24 g (89%).

¹ H NMR (DMSO-d₆, δ): 1.2 (t, 2H); 1.3-1.7 (m, 5H); 2.1 (t, 2H); 2.6(dd, 2H); 2.8 (m, 4H); 3.1 (m, 4H); 4.2 (m, 1H); 4.4 (m, 1H); 6.4 (d,2H); 7.8 (t, 1H); 8.0 (m, 1H).

Reaction Step D: TFP ester of Biotin-caproic acid (48) (0.67 mmol, 0.35g) was dissolved in DMF (40 mL). Triethylamine (80 μL) was addedfollowed by aminoisophthalic acid (1.005 mmol, 0.182 g). The reactionwas stirred at room temp. for 8 days (HPLC monitored) while addingtriethylamine (80 μL) every after 24 h. It was then evaporated todryness. The residue was then applied to a column of silica and wasinitially eluted with acetonitrile (450 mL). It was then eluted withmethanol, 20 mL of fractions were collected, at the fraction 2 thesolvent was changed to DMF. The fractions containing the final product(HPLC monitored) were evaporated to dryness (49) to yield 230 mg (65%).

¹ H NMR (DMSO-d₆, δ): 1.3-1.7 (m, 8H); 2.1 (t, 2H); 2.3 (t, 2H); 2.6 (m,2H); 2.8 (m, 2H); 3.1 (m, 3H); 4.1 (m, 1H); 4.3 (m, 1H); 6.4 (d, 2H);7.8 (t, 1H); 8.1 (m, 1H); 8.46 (s, 2H).

Reaction Step E: Biotin-caproic acid-isophthalic acid (49) (0.376 mmol,200 mg) was dissolved in DMF (30 mL) under argon atmosphere. TFP acetate(0.94 mmol, 241 mg) was added by double ended needle, followed bytriethylamine (112 μL). The reaction was then stirred at room temp. for24 h (HPLC monitored). It was then evaporated to dryness. The lightbrownish oil was taken in ether, solid was filtered and was washed withether (50 mL) (50) to yield 250 mg (86%).

¹ H NMR (DMSO-d₆, δ): 1.3-1.7 (m, 8H); 2.1 (t, 2H); 2.3 (t, 2H); 2.6 (m,2H); 2.8 (m, 2H); 3.1 (m, 3H); 4.2 (m, 1H); 4.4 (m, 1H); 6.4 (d, 2H);7.8 (t, 1H); 8.1 (m, 2H); 8.57 (s, 1H); 8.9 (s, 2H).

Reaction Step F: In a solution of cyanocobalamin carboxylicacid--diaminododecane conjugate (8, 9, 10) (0.130 mmol, 0.2 g) in amixture of DMF:H₂ O (3:1) (40 mL) triethylamine (12 μL) was added. DiTFPester of biotin-caproic acid-isophthalic acid (50) (0.065 mmol, 0.050 g)was added over a period of 5-10 min. The reaction mixture was stirred atroom temperature for 3 h (HPLC monitored). It was then evaporated todryness. The residue was digested with 100 mL of acetone and the solventwas decanted to yield 230 mg (62%) (51). mp 195°-198° C. withdecomposition.

Example 16 Cyanocobalamin Monocarboxylic Acid Diaminododecane ConjugateDimer: Isophthalate Cross-Linking with Para-Iodobenzoyl Moiety

This is an example of a bivalent receptor modulating agent which is alsoconjugated to apara-iodobenzoyl moiety.

Reaction Step A: A 5 g (28 mmol) quantity of 5-aminoisophthalic acid(52) was dissolved in 30 mL 1N NaOH and placed in an ice/water bath. Tothe cold solution was added 7.5 g (28 mmol) 4-iodobenzoyl chloride (52)in 60 mL of acetonitrile, dropwise. The thick white precipitate was thenstirred for 10 minutes before removing the ice/water bath and allowingthe mixture to stir an additional 10 minutes. The reaction mixture wasadjusted to pH 4 with acetic acid and the resulting solid collected.This solid was then dissolved in 30 mL 1N NaOH and washed with ether(2×50 mL). The resulting aqueous solution was filtered and acidified topH 4 with acetic acid. The white precipitate was the collected and driedon high vacuum to yield .6 g (99+% ) of (54). mp >300° C.; IR (Nujol,cm⁻¹) 3570(m), 3300(m), 1645, 1580(m), 1525(m), 760(m); ¹ H NMR(DMSO-d₆, δ), 8.51 (2H, d, J=0.7 Hz), 8.27 (1H, s), 7.94 (2H, d, J=4.2Hz), 7.84 (2H, d, J=4.1 Hz).

Reaction Step B: A 5 g (12.2 mmol) quantity of 5-N-iodobenzoyl)amino!-isophthalic acid (54) was suspended in 100 mLanhydrous ethyl acetate. To this was added 12.5g (73 mmol)2,3,5,6-tetrafluorophenol (55) followed by 5 g (24.2 mmol)1,3-dicyclohexylcarbodiimide. This suspension was then stirred at roomtemperature for 3 days before filtering off the solid and washing withan additional 20 mL of ethyl acetate. The filtrate was then evaporatedto dryness. The resulting sticky white solid was suspended in 50 mLacetonitrile and stirred for 30 minutes. Filtering yielded 3.75g ofwhite solid (43%) (56). mp 250°-251° C.; IR (Nujol, cm⁻¹) 3220(m),3060(m), 1750, 1655, 1520, 1485, 1330, 1195, 1110, 1085, 955(m), 945(m);¹ H NMR (DMSO-d₆, δ), 9.06 (2H, d, J=0.7 Hz), 8.57 (1H, t, J=1.4 Hz),8.04 (2H, m), 7.94 (2H, d, J=4.2 Hz), 7.81 (2H, d, J=4.3 Hz).

Reaction Step C: To a solution of cyanocobalamin carboxylicacid-diaminododecane conjugate (56) (0.192 mmol, 0.3 g) in a mixture ofDMF:H₂ O (3:1) (40 mL) was added triethylamine (0.018 mL). To thissolution, DiTFP ester of5- N-(p-Iodobenzoyl)amino!-Isophthalic acid(57)(0.096 mmol, 0.068 g) was added over a period of 5-10 min. Thereaction mixture was stirred at room temperature for 4-5 h (HPLCmonitored). It was then evaporated to dryness. The solid residue wasdissolved in 20 mL of methanol:H₂ O (8:2) and applied to a reverse phaseC-18 column (500 mm×25 mm, Alltech, 150 psi) which was developed withthe same solvent. RAININ Rabbit-plus peristaltic pumping system was usedwith a DYNAMAX (model UV-1) UV visible absorbance detector; the elutewas collected with an automatic fraction collector. The fractionscontaining the final product (HPLC monitored) were evaporated todryness.

b-acid dimer (58): yield: 280 mg (76%), mp 230°-233° C. withdecomposition, ¹ H NMR (D₂ O, δ) 0.43 (s, 6H, C-20 CH₃); 1.19 (s, 8H);1.3 (m, 36H); 1.37 (d, 12H); 1.46 (s, 10H); 1.63 (m, 8H); 1.87 (s, 12H);2.05 (m, 10H); 2.27 (d, 16H, B10 & B11 CH₃); 2.35 (m, 8H); 2.6 (d, 18H);2.8 (s, 8H); 3.0 (s, 10H); 3.15 (m, 8H); 3.3 (d, 8H); 3.37 (m, 14H); 3.6(m, 2H); 3.68 (d, 2H); 3.76 (m, 2H); 3.9 (d, 2H); 4.07 (m, 2H); 4.12 (m,2H); 4.18 (m, 2H); 4.3 (m, 2H); 4.5 (m, 2H); 4.64 (m, 4H); 6.0 (s, 2H,2C-10); 6.26 (d, 2H, 2R₁); 6.6 (s, 2H, 2B4); 7.1 (s, 2H, 2B2); 7.25 (s,2H, 2B7); 7.7 (d, 2H); 7.9 (d, 2H); 7.99 (d, 1H); 8.28 (s, 2H); MS(FAB⁺): m/e 3453. IR (KBr): 3400, 3200, 2950, 2060, 1660, 1570, 1490,1060 cm⁻¹. UV (MeOH): λ360.6 (ε48 871)

e-acid dimer (59): yield: 258 mg (70%), mp 285°-290 ° C. withdecomposition, ¹ H NMR (D₂ O, δ) 0.43 (s, 6H, C-20 CH₃); 1.17 (s, 8H);1.22 (d, 13H); 1.29 (s, 45H); 1.36 (d, 22H); 1.44 (s, 10H); 1.6 (m, 8H);1.86 (s, 12H); 2.04 (m, 10H); 2.25 (s, 12H, B10 & B11 CH₃); 2.36 (m,8H); 2.55 (d, 20H); 2.83 (m, 8H); 3.15 (m, 8H); 3.29 (s, 10H); 3.36 (m,8H); 3.58 (m, 2H); 3.65 (m, 2H); 3.75 (m, 2H); 3.9 (d, 2H); 4.06 (m,2H); 4.12 (m, 2H); 4.16 (m, 2H); 4.3 (m, 2H); 4.5 (m, 2H); 4.57 (s, 2H);4.65 (m, 2H); 6.0 (s, 2H, 2C-10); 6.26 (d, 2H, 2R1); 6.5 (s, 2H, 2B4);7.1 (s, 2H, 2B2); 7.25 (s, 2H, 2B7); 7.7 (d, 2H); 7.89 (d, 2H); 7.98 (s,1H); 8.26 (s, 2H); MS (FAB⁺): m/e 3453. IR (KBr): 3400, 3200, 2950,2060, 1660, 1570, 1490,1060 cm⁻¹ ; UV(MeOH): λ360 (ε41481).

d-acid dimer (60): yield 265 mg (72%), mp 253°-255 ° C. withdecomposition, ¹ H NMR (D₂ O, δ) 0.43 (s, 6H, C-20 CH₃); 1.16 (s, 8H);1.22 (d, 12H); 1.33 (m, 36H); 1.43 (s, 10H); 1.53 (m, 6H); 1.6 (m, 8H);1.86 (s, 12H); 2.03 (m, 8H); 2.25 (d, 12H, B10 & B11 CH₃); 2.33 (m, 8H);2.54 (d, 20H); 2.8 (s, 4H); 3.0 (s, 4H); 3.28 (s, 10H); 3.35 (m, 8H);3.58 (m, 2H); 3.65 (m, 2H); 3.73 (m, 2H); 3.88 (d, 2H); 4.05 (m, 2H);4.1 (m, 2H); 4.17 (m, 2H); 4.3 (m, 2H); 4.5 (m, 2H); 4.57 (s, 2H); 4.63(m, 2H); 6.0 (s, 2H, 2C-10); 6.26 (d,2H, 2R₁); 6.5 (s,2H, 2B4); 7.1 (s,2H, 2B2); 7.25 (s, 2H, 2B7); 7.7 (d, 2H); 7.89 (d, 2H); 7.98 (s, 1H);8.26 (s, 2H); MS (FAB⁺): m/e 3453. IR (KBr): 3400, 3200, 2950, 2060,1660, 1570, 1490, 1060 cm⁻¹ ; UV (MeOH): λ360 (ε48 245).

Example 17 Cyanocobalamin Monocarboxylic Acid Diaminododecane ConjugateDimer: Isophtahate Cross-Linking with Para-(Tri-Butylstannyl)BenzoylMoiety

This is an example of a bivalent receptor modulating agent coupled to apara-tri-N-butyl stannyl moiety.

Reaction Step A: A 2 g (2.8 mmol) quantity of the diTFP ester of 5-N-(p-Iodobenzoyl)amino!-Isophthalic acid (57) (as prepared above) wasdissolved in 20 mL dry toluene under argon. To this was added 2.8 mL(5.5 mmol) of bis(tributyltin) (61) followed by 40 mg (0.04 mmol)tetrakis(triphenylphosphine)palladium (62). The mixture was stirred atroom temperature for 15 minutes before heating to 80° C. for 2 h. Sincethe mixture only darkened slightly over the 2 h period, an additional 40mg of palladium catalyst was added. Within 1 hour the mixture had turnedblack. After cooling to room temperature, the toluene was removed byrotary evaporation. The resulting black oil (containing solids), wasthen taken into 20 mL ethyl acetate and dried onto 10 g silica gel (viarotoevaporation). This solid was then added to a 250 g (40×3.5 cm)silica gel column and eluted initially with hexanes containing 5% aceticacid. After 600 mL, the solvent was changed to 90/10 hexanes/ethylacetate (containing 5% acetic acid). Fractions 14-16 were combined anddried to yield 1.5 g (62%) of white solid (62). mp 120°-123 ° C.;

¹ H NMR (CDCl₃, δ), 8.87 (2H, d, J=0.7 Hz), 8.76 (1H, t, J=1.6 Hz), 8.38(1H, s), 7.84 (2H, d, J=4.1 Hz), 7.62 (2H, d, J=4.1 Hz), 7.07 (2H, m),1.55 (6H, m), 1.36 (15H,m), 1.11 (6H,m), 0.89 (9H, t, J=7.3 Hz); MS(FAB⁺) M+H patterns calculated 870 (75.1%), 871 (52.9%), 872 (100%), 873(41.0%), 874 (21.4%), found 870 (82.1%), 871 (55.1%), 872 (100%), 873(42.1%), 874 (25.2%).

IR(Nujol,cm⁻¹) 1750, 1645, 1520, 1480(m), 1185, 1100, 1085.

Reaction Step B: In a solution of cyanocobalamin carboxylicacid-diaminododecane conjugate (8, 9, 10) (0.065 mmol, 0.1 g) in amixture of DMF:H₂ O (3:1) (40 mL) triethylamine (0.006 mL) was added.DiTFP ester of 5- N-(p-tributyltin benzoyl) amino!-Isophthalic acid(63)(0.0325 mmol, 0.028 g) was added over a period of 5-10 min. Thereaction mixture was stirred at room temperature for 12-14 h (HPLCmonitored). It was then evaporated to dryness. The residue was digestedwith 100 mL of acetone and the solvent was decanted.

b-acid dimer (64): yield: 90 mg (70%), mp 208°-212 ° C. withdecomposition, ¹ H NMR (D₂ O, δ) 0.43 (s, 6H, C-20 CH₃); 0.88 (t, 9H);1.15 (t, 12H); 1.19 (s, 8H); 1.3 (m, 36H); 1.37 (d, 12H); 1.46 (s, 1OH);1.6 (m, 8H); 1.9 (s, 12H); 2.05 (m, 10H); 2.28 (d, 16H, B10 & B11 CH₃);2.35 (m, 8H); 2.6 (d, 18H); 2.8-2.9 (m, 16H); 3.15 (m, 8H); 3.3 (s, 8H);3.37 (m, 14H); 3.6 (m, 4H); 3.76 (m, 2H); 3.9 (d, 2H); 4.07 (m, 2H);4.12 (m, 2H); 4.18 (m, 2H); 4.3 (m, 2H); 4.5 (m, 2H); 4.68 (m, 2H); 6.0(s, 2H, 2C-10); 6.26 (d,2H, 2R₁); 6.6 (s, 2H, 2B4); 7.1 (s, 2H, 2B2);7.25 (d, 2H, 2B7); 7.6 (d, 2H); 7.9 (d, 2H); 7.99 (br s, 1H); 8.28 (brs, 2H); IR (KBr): 3400, 3200, 2950, 2060, 1660, 1570, 1490, 1060 cm⁻¹.

e-acid dimer (65): yield: 93 mg (72%), mp>300° C., ¹ H NMR (D₂ O, δ)0.43 (s, 6H, C-20 CH₃); 0.88 (t, 9H); 1.12 (t, 12H); 1.17 (d, 8H); 1.22(d, 13H); 1.29 (s, 45H); 1.36 (d, 22H); 1.44 (s, 10H); 1.6 (m, 8H); 1.87(d, 12H); 2.04 (m, 10H); 2.25 (s, 12H, B10 & B11 CH₃); 2.36 (m, 8H);2.55 (d, 20H); 2.8 (m, 8H); 3.15 (m, 8H); 3.29 (s, 10H); 3.36 (m, 14H);3.6 (m, 4H); 3.73 (m, 2H); 3.9 (d, 2H); 4.07 (m, 2H); 4.12 (m, 2H); 4.16(m, 2H); 4.3 (m, 2H); 4.5 (m, 2H); 4.66 (m, 2H); 6.0 (s, 2H, 2C-10);6.26 (d,2H, 2R₁); 6.6 (s,2H, 2B4); 7.1 (s, 2H, 2B2); 7.25 (s, 2H, 2B7);7.6 (d, 2H); 7.9 (d, 2H); 7.98 (br s, 1H); 8.28 (br s, 2H); IR (KBr):3400, 3200, 2950, 2060, 1660, 1570, 1490, 1060 cm⁻¹.

d-acid dimer (66): yield: 100 mg (78%), mp 202°-205 ° C. withdecomposition, ¹ H NMR (D₂ O, δ) 0.43 (s, 6H, C-20 CH₃); 0.88 (t, 9H);1.12 (t, 12H); 1.15 (s, 8H); 1.29 (m, 36H); 1.35 (d, 12H); 1.44 (s,10H); 1.53 (m, 6H); 1.6 (m, 8H); 1.86 (d, 12H); 2.03 (m, 8H); 2.25 (d,12H, B10 & B11 CH₃); 2.33 (m, 8H); 2.54 (d, 20H); 2.8 (m, 8H); 3.13 (m,8H); 3.28 (s, 10H); 3.35 (m, 10H); 3.6 (m, 4H); 3.73 (m, 2H); 3.9 (d,2H); 4.05 (m, 2H); 4.1 (m, 2H); 4.17 (m, 2H); 4.3 (m, 2H); 4.5 (m, 2H);4.6 (m, 2H); 6.0 (s, 2H, 2C-10); 6.26 (d,2H, 2R1); 6.6 (s,2H, 2B4); 7.1(s, 2H, 2B2); 7.25 (s, 2H, 2B7); 7.6 (d, 2H); 7.9 (d, 2H); 7.98 (br s,1H); 8.28 (br s, 2H); IR (KBr): 3400, 3200, 2950, 2060, 1660, 1570,1490, 1060 cm⁻¹.

Example 18 Evaluation of the Ability of Vitamin B₁₂ Receptor ModulatingAgents to Bind to TCII

This example serves to demonstrate a competitive binding assay suitablefor evaluating the ability of vitamin B₁₂ receptor modulating agents tobind TcII. Binding of the vitamin B₁₂ derivatives to recombinanttranscobalamin II was conducted in picomolar concentrations and thepercent bound ascertained.

In this competitive binding assay, various B₁₂ derivatives, includingvitamin B₁₂ receptor modulating agents, were evaluated for their abilityto bind to TcII relative to radiolabeled B₁₂. Varying concentrations ofeach derivative were incubated with immobilized TcII in the presence ofa constant amount of radiolabeled B₁₂. After incubation for 20 minutesat 37° C., the free radiolabeled B₁₂ was separated from the TcII boundtracer by removal of the supernatant. The radioactivity of thesupernatant solution was then measured to determine the amount of freeradiolabeled B₁₂ present at the end of each competition. By measuringthe amount of free radiolabeled B₁₂ for each competition, the ability ofeach derivative to inhibit radiolabeled B₁₂ binding was determined. Abinding curve was then be constructed for each B₁₂ derivative where theamount of radiolabeled B₁₂ bound (% radiolabel bound) was correlatedwith the concentration of derivative present in the original mixture.The more effective the derivative is in binding to TcII, the lower thepercent bound radiolabeled vitamin B₁₂.

FIG. 22 illustrates the binding curve of Transcobalamin II to thecyanocobalamin monocarboxylic acids produced in Example 1.AD=Cyanocobalamin (1); AL=Cyanocobalamin b-monocarboxylic acid (2);AM=Cyanocobalamin e-monocarboxylic acid (3); and AN=Cyanocobalamind-monocarboxylic acid (4). The d-carboxylate (3) appears to bind nearlyas well as cyanocobalamin. Two samples of vitamin B₁₂ were used, one asa known standard and the other as an unknown.

FIG. 23 illustrates the binding curve of Transcobalamin II to thecyanocobalamin diaminododecane adducts (8, 9, 10) and succinate adduct(13) produced in Example 3 and 4 above. AH=Cyanocobalaminb-monocarboxylic acid conj Diaminododecane (7); AI=Cyanocobalamine-monocarboxylic acid conj Diaminododecane (8); AJ=Cyanocobalamind-monocarboxylic acid conj Diaminododecane (9); AK=Cobalamine-monocarboxylic acid conj Diaminododecane, and AE=CyanocobalaminRibose-Succinate (11). The b-conjugate (17) has the least binding,whereas the e-conjugate (18) has intermediate binding, and thed-conjugate (19) binds quite well. The biotin conjugate attached to theribose site (13) appears to bind very well, as does its precursor aminoderivative (12). The additional compound studied is of unknownstructure, but may have the amine group coordinated with the cobalt atomas the mass spectrum indicates that it has the appropriate mass for (7)minus HCN. It is clear that this unknown compound is not likely to bindTcII.

FIG. 24 illustrates the binding curve of Transcobalamin II to a seriesof vitamin B₁₂ dimers. Dimer X=b-acid dimer with Isophthaloyl dichloride(36); Dimer Y=e-acid dimer with Isophthaloyl dichloride (37); dimerZ=d-acid dimer with Isophthaloyl dichloride (38); Dimer A=b-acid Dimerwith p-Iodo benzoyl Isophthaloyl dichloride (58); Dimer B=e-acid Dimerwith p-Iodo benzoyl Isophthaloyl dichloride (59); and Dimer C=d-acidDimer with p-Iodo benzoyl Isophthaloyl dichloride (60).

FIG. 25 illustrates the binding curve of Transcobalamin II to a seriesof biotinylated vitamin B₁₂ molecules. AA=Cyanocobalaminb-monocarboxylic acid conj Diaminododecane and Biotin (17);AB=Cyanocobalamin e-monocarboxylic acid conj Diaminododecane and Biotin(18); AC=Cyanocobalamin d-monocarboxylic acid conj Diaminododecane andBiotin (19); AF=Cyanocobalamin Ribose-Succinate conj Diaminododecane(13); and AG=Cyanocobalamin Ribose-Succinate conj. Diaminododecane andBiotin (20).

Example 19 Assay for Biological Activity of Vitamin B₁₂ ReceptorModulating Agents

This example serves to demonstrate the use of an assay to ascertainbiological activity of the receptor modulating agents of the presentinvention.

Receptor down-modulation involves a comparison of treatment of a targetcell line such as K562, each sample is treated with vitamin B₁₂ or avitamin B₁₂ receptor modulating agent at 4° C. for 24 hours. Followingthis period, cells of each sample are separated from a vitamin B₁₂ or avitamin B₁₂ receptor modulating agent by centrifugation. The cells arethen washed and resuspended in phosphate buffered saline containing 2 mMEDTA for a brief period of time not to exceed 15 minutes at 4° C. Then,the cells are washed again and returned to a tissue culture medium at 4°C. The tissue culture medium containing TcII and a radiolabeled TcII/B₁₂complex. The time course of TcII/B₁₂ binding to the cell receptor isdetermined by measuring the percent radiolabel bound to the cell at 0,15, 30, 60, 120, and 240 minutes. Those samples exposed to the vitaminB₁₂ receptor modulating agents of the present invention showsignificantly reduced TcII/B₁₂ complex binding compared to cellscultured in vitamin B₁₂. Trypsin treated cells reveal any nonspecificbinding or uptake of the labeled vitamin B₁₂ on or within the cell.

Example 20 Synthesis Of An Anti-Inflammatory Receptor Modulating Agent

The synthetic peptide f-met-leu-phe is equivalent to a bacterial cellwall constituent (Biochem. Soc. Trans. 19:1127-9, 1991; Agents ActionsSuppl. 35:3-8, 1991; Agents Actions Suppl. 35:11-6, 1991; J Immunol.146:975-80, 1991). This peptide is recognized by receptors on PMN whichcan respond by chemotaxis to sites of local inflammation along agradient of the peptide. During inflammation, receptor expression can bedramatically increased by mobilizing receptor from intracellular pools.Non-specific methods used to abrogate this up-regulation also inhibitchemotaxis and presumably the anti-inflammatory reaction associated withlocal inflammation (J. Immunol. 145:2633-8, 1990). The synthesis of areceptor modulation agent useful as an inhibitor of early inflammationis described below.

The peptide f-met-leu-phe-(gly)₃ -leu-0-Me is synthesized using tea-bagmethodology or solid phase peptide synthesis procedures described byMerrifield et al. (Biochemistry 21:5020-31, 1982) and Houghten (Proc.Nat'l. Acad. Sci. (USA) 82:5131-35, 1985), or using a commerciallyavailable automated synthesizer, such as the Applied Biosystems 430 Apeptide synthesizer. The peptide-amide is deprotected in 45%trifluoroacetic acid-51% methylene chloride-2% ethanedithiol-2% anisolefor 20 minutes, and cleaved from the 4-methylbenzhydrylamine resin usingthe Tam-Merrifield low-high HF procedure (J. P. Tam et al., J. Am. Chem.Soc. 105:6442-55, 1983). The peptide is then extracted from the resinusing 0.1M ammonium acetate buffer, pH 8, and is lyophilized. The crudepeptide is purified using reverse phase HPLC on a Vydac C-4 analyticalcolumn (The Separations Group, Hesperia, Calif.), and a linear gradientof 0.5-1.0%/min. from 100% acetonitrile+0.1%v/v trifluoroacetate to 100%acetonitrile+0.1% trifluoroacetate. The HPLC-purified peptide isanalyzed by amino acid analysis (R. L. Heinriksen and S. C. Meredith,Anal. Biochem. 160:65-74, 1984) after gas phase hydrolysis (N. M.Meltzer et al., Anal. Biochem. 160:356-61, 1987). The sequence of thepurified peptide may be confirmed by Edman degradation on a commerciallyavailable sequencer (R. M. Hewick et al., J. Biol. Chem. 15:7990-8005,1981). The peptide amide is converted to an O-methyl ester (i.e.,f-met-leu-phe-(gly)₃ -leu-O-Me) by treatment with dimethylformamide (5g/60 mL with 1.3 equivalents of NaHCO₃ in excess methyl iodide (4equivalents). The mixture is stirred under argon gas at room temperaturefor 40 hours. If required, the peptide is extracted to dryness with 150mL of ethyl acetate. The receptor for modulating agent is used to treatPMN, activated with GM-CSF (to increase expression of fMLP receptors).Loss of binding of biotinylated fMLP is compared on FMLP versus f-MLPreceptor modulating agent treated cells.

Example 21 Synthesis Of A Fusion Protein Receptor Modulating Agent

An EGF receptor modulating agent containing a genetically engineeredfusion protein is hereby described. Briefly, the C-terminus of a DNAsequence encoding EGF, or its receptor binding domain, is ligated byconventional procedures (e.g., using T₄ DNA ligase) to a DNA sequencecorresponding to a GGG spacer. The C-terminus of the EGF-GGG DNAsequence is then fused to the N-terminus of a DNA sequence encoding theconditional, membrane binding peptide KGEAALA(EALA)₄ -EALEALAA.Alternately, peptide-spacer DNA sequences may be synthesized in vitrousing standard oligonucleotide synthesis procedures (see, e.g., U.S.Pat. Nos. 4,500,707 and 4, 668,777). The recombinant EGF peptide DNAsequence is cloned in an E. coli expression vector using conventionalprocedures. E. coli strain HB101 is transformed with the fusedrecombinant DNA sequence and cultured to produce the EGF peptide. Thefusion protein is purified form the transformed E. coil culture bystandard methods, including anti-EGF affinity chromatography. The fusionprotein may be eluted from the affinity matrix using standardtechniques, such as high salt, chaotropic agents, or high or low pH.Loss of EGF receptor is measured by flow cytometry and mouse monoclonalantibody to EGF receptor.

From the foregoing, it will be appreciated that, although specificembodiments of this invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except by the appended claims.

We claim:
 1. A method for modulating a vitamin B₁₂ receptor, comprisingadministering an effective amount of a receptor modulating agent to awarm-blooded animal such that a vitamin B₁₂ receptor is modulated,wherein said receptor modulating agent is a vitamin B₁₂ dimer.
 2. Themethod of claim 1 wherein said vitamin B₁₂ dimer is comprised of a firstand a second vitamin B₁₂ molecule coupled through a coupling siteindependently selected from the group consisting of coupling sites a-g,coupling site h, and coupling site i.
 3. The method of claim 2 whereinsaid first and second vitamin B₁₂ molecules are coupled through acoupling site independently selected from the group consisting of d- ande- coupling sites on said first and said second vitamin B₁₂ molecule. 4.The method of claim 3 wherein at least one of said first and said secondvitamin B₁₂ molecules is a vitamin B₁₂ derivative.
 5. The method ofclaim 2 wherein said first and second B₁₂ molecules are coupled throughat least one linker.
 6. The method of claim 5 wherein said linker is atleast 4 atoms in length.
 7. The method of claim 6 wherein said linker isabout 10 to 55 atoms in length.
 8. The method of claim 7 wherein saidlinker is 35 to 45 atoms in length.
 9. The dimer of claim 5 wherein saidlinker includes at least one amino group.
 10. The dimer of claim 9wherein said linker additionally includes a group selected from thegroup consisting of sulfhydryls and carboxyls.
 11. The dimer of claim 9wherein said linker is selected from the group consisting of adiaminoalkyls, diaminoalkylaryls, diaminoheteroalkyls,diaminoheteroalkylaryls, and diaminoalkanes.
 12. The dimer of claim 9wherein said linker is selected from the group consisting of--NH(CH₂)_(x) NH-- wherein x=2-20.
 13. The dimer of claim 9 wherein saidlinker is selected from the group consisting of --NH(CH₂)_(y) CO--,wherein y=3-12.